Centre for Research in Neuroscience, Montreal General Hospital Research Institute; and Department of Neurology and Neurosurgery and Department of Biology, McGill University, Montreal, Quebec H3G 1A4, Canada
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Buss, Robert R. and
Pierre Drapeau.
Synaptic Drive to Motoneurons During Fictive Swimming in the
Developing Zebrafish.
J. Neurophysiol. 86: 197-210, 2001.
The development of swimming behavior and
the correlated activity patterns recorded in motoneurons during fictive
swimming in paralyzed zebrafish larvae were examined and compared.
Larvae were studied from when they hatch (after 2 days) and are first capable of locomotion to when they are active swimmers capable of
capturing prey (after 4 days). High-speed (500 Hz) video imaging was
used to make a basic behavioral characterization of swimming. At
hatching and up to day 3, the larvae swam infrequently and in an
undirected fashion. They displayed sustained bursts of contractions (`burst swimming') at an average frequency of 60-70 Hz that lasted from several seconds to a minute in duration. By day 4 the swimming had
matured to a more frequent and less erratic "beat-and-glide" mode,
with slower (~35 Hz) beats of contractions for ~200 ms alternating with glides that were twice as long, lasting from just a few cycles to
several minutes overall. In whole cell current-clamp recordings, motoneurons displayed similar excitatory synaptic activity and firing
patterns, corresponding to either fictive burst swimming (day 2-3) or
beat-and-glide swimming (day 4). The resting potentials were similar at
all stages (about 70 mV) and the motoneurons were depolarized (to
about
40 mV) with generally non-overshooting action potentials during
fictive swimming. The frequency of sustained inputs during fictive
burst swimming and of repetitive inputs during fictive beat-and glide
swimming corresponded to the behavioral contraction patterns. Fictive
swimming activity patterns were eliminated by application of glutamate
antagonists (kynurenic acid or 6-cyano-7-nitroquinoxalene-2,3-dione and
DL-2-amino-5-phosphonovaleric acid) and were modified but
maintained in the presence of the glycinergic antagonist strychnine.
The corresponding synaptic currents underlying the synaptic drive to
motoneurons during fictive swimming could be isolated under voltage
clamp and consisted of cationic [glutamatergic postsynaptic currents
(PSCs)] and anionic inputs (glycinergic PSCs). Either sustained or
interrupted patterns of PSCs were observed during fictive burst or
beat-and-glide swimming, respectively. During beat-and-glide swimming,
a tonic inward current and rhythmic glutamatergic PSCs (~35 Hz) were
observed. In contrast, bursts of glycinergic PSCs occurred at a higher
frequency, resulting in a more tonic pattern with little evidence for
synchronized activity. We conclude that a rhythmic glutamatergic
synaptic drive underlies swimming and that a tonic, shunting
glycinergic input acts to more closely match the membrane time constant
to the fast synaptic drive.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The use of
simple model systems, such as the lamprey (Grillner et al. 1991,
1998
; Rovainen 1979
) and frog embryo
(Dale and Kuenzi 1997
; Roberts et al. 1986
,
1998
), has greatly increased the understanding of cellular
aspects of the neural control of vertebrate locomotion. Zebrafish
embryos and larvae share with lampreys and frog embryos the
experimental advantages of a simple motor and sensory system
(Drapeau et al. 1999
) and are a popular vertebrate model
system for a wide range of scientific disciplines (Eisen
1996
; Laale 1977
; Vascotto et al.
1997
). An advantage of zebrafish is that they hold promise for
identifying the genes controlling locomotion during development of the
nervous system (Granato et al. 1996
). An understanding
of the physiology of the zebrafish locomotor system is necessary for
eventually assessing genetic mutations and manipulations in zebrafish,
thus ultimately increasing our understanding of the mammalian nervous system.
Movement depends on the activation of locomotor muscles, which are
under the control of spinal motoneurons. In contrast to the complex
motor system of adult fish, with five types of muscle fibers (de
Graff et al. 1990; van Raamsdonk et al. 1983
)
and four classes of motoneurons (van Raamsdonk et al.
1983
), the organization of the developing zebrafish is far
simpler. Embryos and larvae have only two embryonic forms of muscle
that have similar physiological properties (Buss and Drapeau
2000a
). The motoneuronal pool is similarly reduced in
complexity, with only primary and secondary motoneurons present at the
early larval stages (Myers 1985
). Primary motoneurons
form first in development, are the largest motoneurons, and eventually
branch extensively, making contact with nearly all fibers within their
arborization (de Graff et al. 1990
; Myers et al.
1986
; van Raamsdonk et al. 1983
;
Westerfield et al. 1986
). Primary motoneurons are
generally recruited during fast swimming and the startle response
(Fetcho and O'Malley 1995
; Liu and Westerfield 1988
). Secondary motoneurons form later in development, are
smaller than primary motoneurons, branch less extensively in the
muscle, and contact fewer fibers (de Graff et al. 1990
;
Myers et al. 1986
; van Raamsdonk et al.
1983
; Westerfield et al. 1986
). This simplified motor system, when combined with the transparency of the zebrafish, allows visualization of individual neurons for patch clamping (Drapeau et al. 1999
), making the zebrafish an excellent
model organism for studying the neural control of locomotion.
The purpose of this study was to examine the development of swimming in
zebrafish larvae from when they hatch (after 2 days) and are first
capable of locomotion to when they are active swimmers capable of
capturing prey (after four days). High-speed video imaging was used to
make a basic behavioral characterization of swimming that was then
compared with electrophysiological recordings made from identified
motoneurons during fictive swimming in paralyzed larvae. The synaptic
pharmacology of the network of neurons producing the swimming pattern
was investigated by bath-applying antagonists of the major spinal cord
synaptic neurotransmitters and examining their effects on the membrane
potential changes occurring during fictive swimming. Voltage clamping
was then used to isolate the cationic and anionic synaptic currents
underlying the synaptic drive to motoneurons during fictive swimming.
The findings are discussed in relation to the motoneuronal activity
patterns observed during fictive locomotion in fishes and mammals. This
work has been presented previously in abstract form (Buss and
Drapeau 2000b; Buss et al. 1999
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Swimming behavior
High-speed video (Motionscope 500, Redlake Camera Opticon) was used to analyze the swimming pattern of larval zebrafish aged 2.4-2.8 (n = 11) and 4.9-5.2 (n = 12) days postfertilization (dpf), referred to as day 2 and day 4. Eighteen larvae were placed in a 6-cm plastic petri dish illuminated by overhead halogen lighting. Spontaneously occurring swimming was filmed at 500 frames/s, and frames were stored on VCRT120 tape with a Panasonic AG7300 VCR. Images were captured to computer using NIH/Scion Image software for further viewing and analysis. Average camera jitter was ~3% and was not corrected.
Two distinct forms of swimming were observed in larval zebrafish:
continuous bursts of swimming at day 2 (burst swimming) and an
intermittent style of swimming characterized by tail beating followed
by gliding (beat-and-glide swimming) at day 4. The terminology is taken
from Hunter (1972), who used it to describe the swimming patterns of the larval anchovy. The swimming parameters were related to
those that were measurable in patch-clamped motoneurons during fictive
swimming. The parameters included swim duration, number of tail beats,
tail-beat frequency, distance covered, and duration of either phase of
beat-and-glide swimming. Figures were created by hand-tracing
sequential computer printouts of the high-speed video recordings.
Tracings were scanned and enhanced for display purposes using Adobe Photoshop.
Preparation for recording
Experiments were performed on zebrafish (Danio rerio)
larvae of the Longfin strain raised at ~28.5°C and obtained from a
breeding colony maintained according to Westerfield
(1993). Physiological results are taken from recordings made in
66 morphologically identified (dye-filled) motoneurons. Zebrafish were
examined at three ages: after hatching (2.0-2.8 dpf; referred to as
day 2), a day later in development (3.1-3.4 dpf; referred to as day 3)
and after the onset of active swimming and feeding (4.1-4.5 dpf;
referred to as day 4). All procedures were carried out in compliance
with the guidelines stipulated by the Canadian Council for Animal Care and McGill University as described previously (Drapeau et al. 1999
). Larvae were anesthetized in 0.02% tricaine (MS-222,
Sigma) dissolved in fish saline, pinned through the notochord to a
silicone elastomer (Sylgard)-lined dish, and the skin overlying the
axial musculature removed with a glass pipette and fine forceps. Muscle fibers were removed from one or two myotomal segments in the anal region by aspiration with a broken patch pipette to expose the spinal
cord. Experiments were performed at room temperature (~22°C). The
fish saline resembled the plasma of freshwater fish (Evans 1998
; Heisler 1984
; Holmes and Donaldson
1969
; McDonald and Milligan 1992
) and contained
(in mM) 134 NaCl, 2.9 KCl, 2.1 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose, osmolarity 280 to 290 mOsm and pH 7.8. In most experiments (n = 42),
the neuromuscular blocker
-bungarotoxin (10 µM, Sigma) was added
to the dish for 10-20 min and then replaced with a 0.1% collagenase
(type XII, Sigma) fish saline solution for 8-10 min. The collagenase
was removed, and the preparation was perfused with fish saline for the
remainder of the experiment. When D-tubocurarine (15 µM,
Sigma) was used as a neuromuscular blocker (n = 24), it
was added directly to the fish saline. There was no noticeable
difference in the fictive swimming activity of zebrafish paralyzed with
-bungarotoxin or D-tubocurarine.
Whole cell recordings
Standard whole cell recordings (Hamill et al.
1981) were performed on motoneuron cell bodies visualized with
Hoffman modulation optics (×40 water-immersion objective). Patch-clamp
electrodes (4-7 M
) were pulled from thin-walled Kimax-51
borosilicate glass and were filled with either a potassium gluconate
(for current-clamp recordings) or cesium gluconate solution (for
voltage-clamp recordings). All voltage-clamp recordings were performed
on
-bungarotoxin-paralyzed larvae. The potassium gluconate solution
was composed of (in mM) 116 D-gluconic acid potassium salt,
16 KCl, 2 MgCl2, 10 HEPES, 10 EGTA, 4 Na2ATP, and 0.2% sulforhodamine B, osmolarity
280-290 mOsm, pH adjusted to 7.2. In the cesium gluconate solution,
potassium gluconate and KCl were replaced with cesium gluconate and
CsCl and 0.5-1.0 mM lidocaine N-ethyl bromide
(QX-314) was added to antagonize voltage-activated sodium and
calcium currents. The liquid junction potential was
5 mV, and records
were corrected for this potential. Current-clamp recordings were
performed with an Axoclamp-2A patch-clamp amplifier (0.01 headstage; 10 kHz low-pass filter) and voltage-clamp recordings with an
Axopatch 1D (CV-4 headstage; 5 kHz low-pass filter; series resistance
10 M
compensated 60-80%). The digitization rate was at 20-40
kHz. Each neuron was positively identified as a motoneuron by its
location just dorsal to the central canal and the presence of an axon
exiting the spinal cord and branching throughout the myotomal muscle,
as viewed under fluorescence optics. Images were captured with a
Panasonic BP510 CCD camera and a Scion Corporation LG3 frame grabber
using Scion/NIH Image software. Voltage steps of 20-40 mV elicited
fast, transient inward currents (tested immediately after whole cell
configuration was achieved) in all motoneurons. The QX-314 present in
the pipette abolished these transient currents within 1-2 min.
The motoneurons examined were fully dialyzed with the patch electrode
recording solution since sulforhodamine B fluorescence was detected
throughout the motoneurons including the extensive axonal arborization
in the myotomal muscle. The patch recording solution contained 20 mM
chloride; this, on consideration of the activity coefficient
(Parsons 1959), places the chloride reversal potential
at
46 mV (calculated using the Nernst equation corrected for
Debye-Hückel activities). Thus at the resting potential, glycinergic postsynaptic potentials [e.g., irregular postsynaptic potentials (irregular PSPs)] were easily identified as small
depolarizing PSPs. Buss et al. (1999)
and
Saint-Amant and Drapeau (2000)
have demonstrated that
the chloride ion is depolarizing in vivo in developing zebrafish such
that all postsynaptic potentials are depolarizing at the resting
membrane potential.
Pharmacological antagonists were dissolved in fish saline and applied by bath perfusion. Strychnine hydrochloride (2 µM), kynurenic acid (1 mM), and tetrodotoxin (1 µM) were purchased from Sigma and DL-2-amino-5-phosphonovaleric acid (AP-5, 50 µM) and 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX, 10 µM) from RBI (Natick, MA). Sulforhodamine B was purchased from Molecular Probes (Eugene, OR) and all other chemicals from Sigma Chemical (St. Louis, MO).
Analyses
Analyses were performed using pClamp 6 or Axograph 4.4 (Axon
Instruments). Measurements of fictive swimming rhythmic excitatory postsynaptic potential and current (EPSP and EPSC) frequency or amplitude, tonic depolarization or current, and action potential amplitude or threshold were made by eye (cursor measurement). Rhythmic
EPSCs were detected using the template function of Axograph 4.4. Detected events were divided into two size groups, averaged, and the
decay phase () of the averaged EPSC fit to a single exponential function.
Mean rhythmic EPSC amplitudes and frequencies were determined from consecutive measurements made on the first 30-50 EPSCs occurring in an episode of fictive swimming. EPSP measurements were made on ~10 consecutive EPSPs, which occurred during periods of fictive swimming, where the synaptic drive was sufficient to drive occasionally the membrane to action potential threshold. The action potential threshold values presented are the membrane potential at which rhythmic EPSPs initiated action potential firing. Results are presented as means ± SE throughout the text. The term significant denotes a relationship with P < 0.05 determined using the Student's t-test, Mann-Whitney rank sum test, or paired t-test.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Behavioral characterization of larval swimming
From hatching (after day 2) through day 3, zebrafish are largely inactive, lying on their sides on the substrate. Spontaneous swimming is infrequent and occurs in sustained bouts lasting from a few seconds to less than a minute. Changes in light intensity often evoked episodes of swimming. Figure 1 shows images taken every 10 ms while recording images at 500 Hz. The swimming effectively propels the larvae but is erratic, with many turns and displacements of the head, and lacks a definite direction (Fig. 1A). Tail-beat frequencies are very rapid, approaching 100 Hz in some instances and averaging 67 ± 6 Hz (n = 11). This form of swimming will be referred to as "burst swimming." The kinematics of swimming is similar in day 2 and day 4 larvae in that swimming movements are eel-like (anguilliform) and are characterized by a wave traveling in a rostral to caudal direction (Fig. 1, insets). However, at day 4, the larvae maintained a more constant (forward) orientation. Furthermore the structure of the swimming changed from sustained burst swimming to an intermittent style of swimming (Fig. 1B) where there is a period of active tail-beating and propulsion (beat period) followed by a period of inactivity where the larvae rapidly glide to a stop (glide period). Episodes of beating and gliding could repeat for longer than a minute or could persist for only a few cycles. This form of swimming will be referred to as "beat-and-glide swimming." Beat-and-glide swimming was much less erratic than the earlier burst swimming; larvae made frequent turns, but their movements were more directed, and they could maintain a suspended position in the water column even though their swim bladders were usually not yet functional at this stage of development. The average duration of the tail-beat periods was 180 ± 20 ms (n = 12), and these periods were followed by a period of gliding lasting on average 390 ± 30 ms (Table 1). The mean distance traveled during the tail-beat period was 4.7 ± 0.5 mm, while on average 37 ± 7% of this distance was covered during the glide period. Two to eight tail beats occurred during the beat period (mean = 4.7 ± 0.5), and tail-beat frequencies averaged 35 ± 2 Hz (range = 25-63 Hz).
|
|
Properties of fictive swimming
The preceding behavioral observations revealed that larval
zebrafish spontaneously initiate swimming and that changes in light intensity could induce swimming. As ventral roots are too small and
inaccessible for recording in the zebrafish larva, we resorted to whole
cell patch-clamp recordings from motoneurons to characterize the
cellular and synaptic activity patterns during swimming. Motoneuron activity patterns were examined in paralyzed larval zebrafish with the
expectation that the nervous system would continue to produce rhythmic
activity destined to activate the myotomal locomotor muscle in a way
appropriate for swimming. Current-clamp recordings revealed stable
resting membrane potentials ranging from 60 to
76 mV at all stages
examined (Table 2; mean =
69 ± 1 mV) and small, intermittently occurring (possibly spontaneous)
postsynaptic potentials, which were never large enough to elicit action
potentials (Fig. 2). Periodically, or in
response to changes in illumination, motoneurons depolarized and fired
rhythmic action potentials with a temporal pattern consistent with the
motoneuron output expected for swimming. This activity, believed to be
fictive swimming, was further examined and compared with the
free-swimming behavior.
|
|
The fictive swimming consisted of a tonic depolarization and rhythmic
postsynaptic potentials. At day 2 (Fig. 2A), the tonic depolarization driving the fictive swimming was sustained for tens of
seconds (mean = 11 ± 2 s). In day 4 motoneurons (Fig. 2C), the tonic depolarization lasted only a few hundred
milliseconds and was followed by a repolarization of the membrane
toward the resting potential. At day 4, these periods of depolarization
and repolarization could occur repeatedly often for several minutes or
as long as the recordings (in this case making it impossible to
quantify the duration of beat-and-glide swimming) but sometimes for
only a few cycles. Each of these fictive swimming patterns corresponded
closely to the burst swimming observed in day 2 larvae and the
beat-and-glide swimming observed in day 4 larvae. Day 3 (Fig.
2B) motoneurons behaved similarly to day 2 motoneurons. However, in some recordings, an episode of fictive burst swimming was
followed by activity resembling beat-and-glide fictive swimming although there was an incomplete membrane repolarization during the
glide period. The addition of the beat-and-glide like activity could
prolong the day 3 swimming episodes for 2-3 min (mean = 33 ± 11 s). As day 3 animals developed further, the swimming style became increasingly more like beat-and-glide swimming. However, even at
day 4, short lasting periods of burst like swimming could occur
preceding a much longer period of beat-and-glide fictive swimming.
The swimming parameters examined were related to those that were
measurable in patch-clamped motoneurons during fictive swimming. The
parameters included swim duration, number of tail beats, tail-beat frequency, and duration of either phase of beat-and-glide swimming. Tables 1 and 2 present the values for these and other parameters during
free and fictive swimming. The average membrane potential (Table 2)
reached during the tonic depolarization phase of fictive swimming was
54 ± 1 mV during both fictive burst swimming (day 2-3) and
fictive beat-and-glide swimming (day 4). The rhythmic postsynaptic
potentials (i.e., the network output) could reach action potential
threshold (
41 ± 0.8 mV) at all stages. The action potential
threshold values were measured as the membrane potential at which
rhythmic EPSPs initiated action potential firing. Rhythmic EPSPs during
fictive burst swimming (day 2-3) occurred with a mean frequency of
52 ± 2 Hz, while the frequency during beat-and-glide fictive
swimming was significantly lower (35 ± 3 Hz). All rhythmic EPSPs
did not evoke action potentials, nor did all motoneurons fire action
potentials during fictive swimming, even though a tonic depolarization
and rhythmic EPSPs were clearly visible. Some rhythmic EPSPs could
evoke one to four action potentials on their rising phase in all larval
age groups. Action potentials elicited during fictive swimming reached
an average peak membrane potential of
14 ± 2 mV that was
similar at all stages but could on occasion be overshooting in an
individual motoneuron. Action potentials did not have an afterhyperpolarization.
A smaller PSP, occurring between the rhythmic EPSPs, was observed occasionally, in 20 of the 38 motoneurons examined. When present (Fig. 2, A and B, * in insets), these other PSPs did not occur regularly throughout the fictive swimming episodes. The irregular PSPs were more clearly resolved under voltage clamp (described in the following text). The largest and most clearly defined irregular PSPs usually occurred when the excitatory synaptic drive was greatest during a fictive swimming episode. During periods of weak rhythmic synaptic drive, especially during day 4 beat-and-glide fictive swimming, the irregular PSP was not observed (Fig. 2C, inset).
In day 2 and day 3 larvae, bursts of fictive swimming were sometimes followed by a small 0.5- to 3-mV hyperpolarization that was sustained for 10-40 s (Fig. 2). At day 2, fictive swimming was followed by a 2.1 ± 0.2-mV hyperpolarization lasting 14 ± 3 s in 12 of 15 motoneurons. Five of 16 day 3 motoneurons displayed a hyperpolarization (mean amplitude = 1.3 ± 0.3 mV; mean duration = 17 ± 3 s) following a burst of fictive swimming. Membrane hyperpolarization was not observed following beat-and-glide swimming in day 4 motoneurons (Fig. 2C).
From these recordings it was not possible to determine the nature of
the depolarizing drive to the motoneurons during fictive swimming. The
depolarizing drive appeared synaptic in origin and current pulses
sufficient to depolarize or hyperpolarize the membrane potential 20-40
mV (for tens to hundreds of milliseconds) during episodes of fictive
swimming did not disrupt the following motoneuron output (not shown).
Bath application of TTX (n = 4) abolished fictive
swimming. However, some motoneurons may have intrinsic oscillatory
properties as small (<2 mV), long-lasting (~1 s) membrane depolarizations were observed (3 of 4 motoneurons) in the presence of
TTX (see also Ali et al. 2000a).
Pharmacology of fictive swimming
To gain insight into the pharmacology of the fictive
swimming synaptic drive, receptor antagonists of the major zebrafish spinal cord synaptic neurotransmitters, glutamate (Ali et al. 2000a) and glycine (Ali et al. 2000b
), were bath
applied to the preparation. Addition of the glutamate receptor
antagonist kynurenic acid or a combination of the specific AMPA/kainate
and N-methyl-D-aspartate (NMDA) receptor
antagonists (CNQX and AP-5) to the fish saline abolished spontaneous or
light-induced fictive swimming (n = 4). The resting
membrane potential was unaffected by the glutamatergic antagonists, and
the remaining spontaneous synaptic activity was blocked by the glycine
receptor antagonist strychnine. Cholinergic synaptic drive was not
critical for the production of fictive swimming as no noticeable
difference in motoneuron activity was observed if
-bungarotoxin or
D-tubocurarine was used to paralyze the preparations.
In contrast, blocking glycinergic transmission (n = 6)
by bath application of strychnine did not abolish rhythmic activity (Fig. 3) even though it causes spasms of
bilateral contractions in intact larvae (Granato et al.
1996). Strychnine did not significantly affect the frequency of
rhythmic EPSPs (strychnine =
54 ± 2 Hz, control =
51 ± 2 Hz) or the tonic synaptic drive (
53 ± 2 vs.
53 ± 2 mV) during a fictive swimming episode observed in single motoneurons. There was an abolition of the irregular PSP and a distinct
increase in motoneuron spiking during fictive swimming. Significantly
more action potentials occurred per second of fictive swimming
(6.5 ± 2 vs. 2.4 ± 1); this was attributable to the
initiation of an extra one or two action potentials by many of the
rhythmic EPSPs. Action potential threshold significantly decreased by 5 mV (
40 ± 2 vs.
45 ± 1 mV, P = 0.019),
and action potential height significantly decreased by 13 mV (
25 ± 4 vs.
12 ± 2 mV) in the presence of strychnine. Fictive
swimming duration and resting membrane potential were not noticeably
affected by strychnine, while a hyperpolarization following fictive
swimming was revealed in two cells that did not display it prior to
strychnine application.
|
Properties of fictive swimming synaptic drive
The preceding current-clamp recordings revealed that motoneurons
depolarized in both a phasic and tonic pattern during fictive swimming,
although the nature of this depolarization was not positively identified. Bath application of glutamatergic and glycinergic antagonists either abolished or changed, respectively, the fictive swimming motor pattern, a result consistent with a glutamatergic and
glycinergic synaptic drive underlying the fictive swimming depolarization. However, bath-applied antagonists do not act solely on
motoneurons, and the changes observed could be due to indirect actions
on other neurons active during fictive swimming. To overcome this
shortfall in the current-clamp analysis of motoneuron activity patterns, motoneurons were voltage clamped at the reversal potential for chloride ion to reveal isolated cation currents (presumably glutamatergic) or at the cation reversal potential to reveal isolated chloride ion currents (presumably glycinergic) without pharmacological perturbation of network activity. Furthermore due to the small size of
larval zebrafish neurons, an effective space clamp is achieved for
synaptic currents (Ali et al. 2000a,b
; Drapeau et al. 1999
), allowing for a more quantitative index of synaptic activity.
Motoneurons that were voltage clamped at the chloride reversal
potential displayed spontaneous or light-evoked bursts of inward synaptic currents composed of a tonic inward current and rhythmic EPSCs
(Fig. 4). The chloride ion reversal
potential was set in each motoneuron by determining the reversal
potential of the spontaneously occurring glycinergic synaptic currents,
i.e., the reversal potential was not calculated but determined
experimentally (about 42 mV). In general, the same frequency of
activity was observed in voltage-clamped motoneurons as was observed in
the current-clamped motoneurons (Table 2). Day 2 and 3 motoneurons
(Fig. 4, A and B) displayed activities consistent
with the motor output required to produce burst swimming and day 4 motoneurons (Fig. 4C) that required to produce
beat-and-glide swimming. These findings indicated that the depolarizing
drive underlying fictive swimming was synaptic in nature and that the
rhythmicity producing the motor output was due to a cationic
conductance (presumably glutamatergic). The tonic inward current
averaged 41 ± 6 pA and tended to vary from cell to cell but did
not change significantly with development (Table 2). Rhythmic EPSC
amplitudes sometimes exceeded 200 pA (mean = 49 ± 6 pA) and
increased significantly with development (Table 2). The frequency of
the rhythmic EPSCs (Table 2) were higher during day 2 and 3 fictive
burst swimming (mean = 45 ± 2 Hz) than during the fictive
beat-and-glide swimming (mean = 37 ± 3 Hz).
|
If the rhythmic EPSCs were glutamatergic, they should have kinetic
characteristics resembling those of the spontaneous, quantal glutamatergic synaptic currents previously characterized in zebrafish motoneurons (Ali et al. 2000a). That study revealed
biexponential decay time constants for both AMPA/kainate (faster
= 0.5-0.8 ms, slower
= 3-6 ms) and NMDA (faster
= 5-8 ms, slower
= 30-45 ms) components of
spontaneous synaptic currents. To examine the possible contributions of
these two types of glutamate receptors, decays were fit to a single
exponential function to approximate roughly the time course of decay of
the rhythmic EPSCs. Fits to more complex time courses were not possible
due to interruption of the summated evoked events. Rhythmic EPSCs
varied in amplitude and the largest EPSCs appeared to have a faster
rate of decay. To verify this apparent difference, the rhythmic EPSCs
were divided into two size groups containing small (10-25 pA) or large
(>50 pA) EPSCs. The decay time course of the rhythmic EPSCs varied from 2.8 ms (day 2 large EPSCs) to 5.9 ms (day 4 small EPSCs). The time
course of the large EPSCs was faster than that of the small EPSCs at
all stages examined, and this difference was statistically significant
in day 2 and 3 motoneurons (Table 3).
Furthermore, in low-noise, high-resolution recordings (Fig.
6C), the rhythmic EPSCs were found to be composed of many
small currents that closely resembled the miniature AMPA/kainate
synaptic currents described by Ali et al. (2000a)
.
|
The preceding current-clamp recordings revealed the presence of an
occasional small, irregular PSP at days 2 and 3 that was presumably a
glycinergic chloride ion conductance as it was not observed in the
presence of strychnine. To gain a greater understanding of this PSP,
motoneurons were voltage clamped at a potential (about 28 mV)
intermediate to the chloride reversal potential and the cation reversal
potential to reveal a mixture of inward cation currents and outward
chloride ion currents.
At the intermediate holding potential, rhythmic inward currents were
superimposed on a sustained tonic chloride ion current. This was
observed in day 2-3 motoneurons (Fig.
5B) displaying fictive burst
swimming as well as in day 4 motoneurons (Fig.
6B) displaying fictive
beat-and-glide swimming. To study the chloride current in isolation,
the holding potential was depolarized further to ~5 mV, the cation
reversal potential (Ali et al. 2000a). At this
potential, it was readily apparent that there was mostly a sustained,
tonic chloride current occurring during fictive swimming (Figs.
5A and 6A). Closer examination revealed that the
chloride current appeared to be composed of many individual synaptic
currents that closely resembled the glycinergic synaptic currents
described in larval zebrafish motoneurons (Buss et al.
1999
) and reticulospinal neurons (Ali et al.
2000b
). The peaks of these synaptic currents showed
rhythmicity, which was likely to account for the occasional irregular
PSPs observed in the current-clamp recordings. Because it was
difficult to voltage clamp motoneurons at such a depolarized level, the
rhythmicity of the chloride synaptic current peaks was only
examined in 11 cells (day 2 = 4; day 3 = 3; day 4 = 4).
|
|
Closer examination of the chloride ion PSCs revealed that they did not always display (Fig. 5A, inset, vs. 6A) the same high degree of rhythmicity as the rhythmic cationic EPSCs described in the preceding text. The timing of the chloride ion PSC peaks varied greatly, and during a single episode of fictive swimming inter-peak frequencies could range from 20 to 500 Hz. Although the rhythmic cationic EPSCs were invisible at this potential, it was reasonable to conclude that some of the chloride current peaks would be occurring midway between the rhythmic cationic EPSCs. However, many of these chloride current peaks could also be occurring synchronously with rhythmic cationic EPSCs. The frequency of the chloride current peaks was measured and normalized, on a cell-by-cell basis, to the frequency of the rhythmic cationic EPSCs believed to underlie the timing of tail beats in the free swimming larvae. On average, the chloride current peaks occurred at a frequency two to three times higher than the rhythmic cationic EPSCs (2.6 ± 0.2 times, day 2; 2.0 ± 0.5 times, day 3; 2.7 ± 0.6 times, day 4). This finding supports the occurrence of rhythmic synaptic chloride current peaks (irregular PSPs) occurring with rhythmic cationic EPSCs. However, the additional conductance added by the peak of the chloride current was small compared with the magnitude of the tonic chloride conductance occurring during fictive swimming, and the physiological significance of the chloride current most likely lies in its tonic component. Noteworthy is the fact that the system-wide antagonism of glycinergic currents did not disrupt rhythmic synaptic activity, while it had clear affects on the firing properties of the motoneuron during this activity.
Comparison of fictive swimming and free swimming
Motoneurons displayed the same fictive swimming activity whether they were examined using current- or voltage-clamp recording techniques (Figs. 2 vs. 4-6). The measured parameters were not significantly different so all fictive swimming data were pooled and compared with the parameters measured in free swimming larvae. In both fictive and free swimming, tail-beat/rhythmic EPSP frequency was higher during burst swimming than during beat-and-glide swimming (Table 2). The mean tail-beat/rhythmic EPSP frequency during beat-and-glide swimming was identical (35 Hz) during fictive and free swimming, although during burst swimming it was significantly higher during the behavior than during fictive swimming (Table 1). During day 4 beat-and-glide swimming (Tables 1 and 2), the mean beat periods were very similar, while the mean glide periods were significantly longer during fictive swimming. Although there were differences in the preceding mean values, all values recorded during fictive swimming were within the range of values observed in the free swimming behavior. The higher observed free swimming values could be due to the presence of active sensory feedback during free swimming or could simply be because the most active (and fastest swimming) larvae swam through the field of view during the high speed video recording. Another difference between free and fictive beat-and-glide swimming was the occurrence of about two fewer tail beats per beat period (when compared with the mean number of rhythmic EPSPs/EPSCs) occurring during fictive swimming (Table 1). This difference is likely because of the inclusion of small sub-threshold EPSPs/EPSCs when calculating the number of rhythmic EPSPs (i.e., fictive tail beats) in the fictive beat period. The addition of these sub-threshold EPSPs would also explain the longer mean burst periods observed during fictive beat-and-glide swimming (Table 1).
Dye coupling between motoneurons and other neurons
Dye coupling between the patched motoneuron and the axon of another neuron was clearly observed in 3 of the 66 dye-filled motoneurons examined in this study. In two instances, the dye-coupled axon could be traced to a dye-filled cell body that was always an ipsilateral descending interneuron. Whether the other dye-filled axon was a descending interneuron, propriospinal neuron, or descending axon from the brain stem could not be determined. Dye coupling between motoneurons and other motoneurons was never observed.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Free swimming
Beat-and-glide swimming has been previously examined in larval
zebrafish (Budick and O'Malley 2000; Fuiman and
Webb 1988
) while the transition from burst swimming to
beat-and-glide swimming has not. In addition, Saint-Amant and
Drapeau (1998)
have described the earliest embryonic swimming.
From the onset of swimming at 28-36 h, a period when the embryo
remains encapsulated in the egg, the tail-beat frequency increases from
7 to 27 Hz. The present study shows a continued twofold increase in
tail-beat frequency during the next day of development.
The mean tail-beat period (180 ms) and glide period (390 ms) described
in this study matched closely with the 130-ms tail-beat period and the
range of glide periods (400-2,000 ms, which average toward the lower
range values) described by Fuiman and Webb (1988). As
described in this other study, episodes of beat-and-glide swimming regularly began with a turn and swimming in a new direction. The mean
tail-beat frequency observed in this study (35 Hz) corresponds to the
spontaneously initiated swimming described by Budick and O'Malley (2000)
. A similar transition from burst swimming to
beat-and-glide swimming has been observed in anchovies by Hunter
(1972)
. Similar to zebrafish, at hatching, anchovies remain
motionless except for brief (1-2 s) spontaneous bursts of swimming
characterized by continuous tail beating at rates
50 Hz. Within a few
days, a new dominant mode of intermittent swimming emerges consisting of alternating periods of swimming and gliding using lower tail-beat frequencies. For hydrodynamic reasons, it is advantageous for small
larval fish to swim continuously and rapidly as gliding is not
physically possible. Larger larval and adult fish swim and glide, which
is then possible due to their larger, more streamlined shape
(Webb and Weihs 1986
; Weihs 1980
). Much
later in development, the anguilliform swimming of larval zebrafish
(the common mode of swimming in larval fish) is replaced by the
subcaragiform mode of the adults, which is characterized by a reduced
side-to-side eel-like motion in the anterior end of the fish
(Lindsey 1978
).
Fictive swimming
The spontaneous or light-evoked episodes of depolarization and rhythmic action potential firing in motoneurons are consistent with a fictive motor pattern that would activate myotomal muscle in a pattern appropriate for swimming in a non-paralyzed preparation. The frequency of rhythmic EPSPs, EPSCs, and action-potential firing recorded in paralyzed preparations closely matched the free swimming tail-beat frequencies (Tables 1 and 2). A developmental change from burst to beat-and-glide swimming was similarly observed in the free swimming and paralyzed preparations, and the underlying structure of the beat-and-glide swimming was similar.
A tonic depolarization and rhythmic EPSPs capable of initiating action
potentials characterize fictive swimming in day 2-4 zebrafish. The
tonic depolarization arises from cationic synaptic currents, which
likely sum with tonic chloride ion synaptic currents, due to the
depolarizing nature of chloride ions in these developing motoneurons
(Buss et al. 1999; Saint-Amant and Drapeau
2000
). Synaptic cation currents are hypothesized to be
glutamatergic based on four observations: glutamatergic antagonists
abolished fictive swimming, glutamatergic antagonists abolished all
cationic miniature EPSCs in zebrafish motoneurons (Ali et al.
2000a
), summing synaptic currents with the properties of
AMPA/kainate mEPSCs were observed in the rhythmic EPSCs, and
cholinergic synaptic currents were never observed in larval zebrafish
motoneurons (Buss and Drapeau 2000b
).
We hypothesize that the rhythmic EPSPs are formed by fast ( = 0.5-0.8 ms and 3-6 ms) AMPA/kainate synaptic currents, combined with
the faster component (with
= 5-8 ms) of the NMDA synaptic currents (Ali et al. 2000a
). Table 3 shows that the
decay time constant of the largest rhythmic EPSCs (~3 ms) is faster
than the faster decay time constant of NMDA synaptic currents. This indicates that the faster AMPA/kainate channels could carry much of
this current. The smaller rhythmic EPSC currents (~6 ms) are close to
the value of the slow time constant of AMPA/kainate synaptic currents
as well as the fast time constant of NMDA synaptic currents and may
thus be due to a combination of inputs from these receptors. The
prolonged decay time course of NMDA synaptic currents (slower
= 30-45 ms) (Ali et al. 2000a
) arising from either
mixed NMDA/AMPA synapses or pure NMDA synapses, could provide much of
the sustained tonic depolarization. However, an additional tonic drive
mediated by slow acting metabotropic glutamate receptor or muscarinic
cholinergic receptor activated channels cannot be ruled out. The
glutamatergic synaptic transmission is mediated by
action-potential-evoked synaptic release as fictive swimming was
abolished by TTX application.
Chloride-mediated, glycinergic synaptic currents occurred concurrently
with the glutamatergic currents. These chloride-ion-mediated synaptic
currents were concluded to be glycinergic based on three observations:
strychnine abolished the irregular PSP, glycinergic antagonists
abolished all chloride ion mediated mPSCs (except for rare,
infrequently occurring bicuculline-sensitive GABAergic mPSCs observed
in a small percentage of motoneurons) in larval zebrafish motoneurons
(Buss et al. 1999) and resticulospinal neurons (Ali et al. 2000b
), and the decay time course and
appearance of the chloride mediated synaptic currents observed during
fictive swimming resembled glycinergic synaptic currents described by Buss et al. (1999)
and Ali et al.
(2000b)
.
The peaks of these synaptic chloride currents formed the irregular PSPs, which were present in approximately half of the motoneurons examined but represented only a third of all the peaks observed. Moreover the chloride currents were largely tonic in nature and were not essential to the patterning of the locomotor rhythm since eliminating glycinergic synaptic currents with strychnine had no significant affect on the frequency of the rhythmic EPSPs. However, strychnine did affect motor output, causing a significant increase in the frequency of action potentials during fictive swimming as well as a decrease in action potential amplitude and threshold.
The tonic chloride conductance occurring during fictive swimming could act to decrease the input resistance and consequently the membrane length and time constants of the motoneurons. Strychnine caused a decrease in action potential threshold and amplitude, and these effects may reflect an increased motoneuron input resistance, length, and time constants. As the action potential of larval zebrafish motoneurons occur during the decay of the membrane depolarization evoked by short (2 ms) current injections (unpublished observations), the spikes are likely initiated in the axon and not the soma. A longer membrane length constant, after application of strychnine, would lessen the attenuation of the membrane depolarization from the spike initiation zone to the soma, resulting in a perceived lowering of the action potential initiation threshold measured at the soma.
Blocking the chloride conductance with strychnine would increase the
membrane time constant and reduce the recording bandwidth, resulting in
filtered action potentials of smaller amplitude. Shortening of the
membrane time constant due to a sustained, glycinergic chloride
conductance will serve to shorten the time course of synaptic
potentials. Larval zebrafish motoneurons have input resistances an
order of magnitude larger than reported in adult fish but have to swim
with much faster undulations to propel themselves through the water.
Glutamatergic synaptic currents (AMPA/kainate) have very fast kinetics
(0.5-0.8 ms) (Ali et al. 2000a) and motoneurons produce
a coordinated rhythmic synaptic output sometimes reaching 100 Hz during swimming.
Electrical transmission
Although the sources of synaptic drive to motoneurons during
fictive swimming are attributed to chemical synapses (glutamatergic and
glycinergic), it is probable given the results of dye-coupling experiments that electrical synapses provide an additional source of
synaptic drive. In the zebrafish embryo, electrical transmission appears to play a critical role in the production of the spontaneous motor activity occurring during the first day of development prior to
the appearance of chemical synaptic transmission in embryonic motoneurons (Saint-Amant and Drapeau 2000). Electrical
synapses have been extensively examined in adult fishes as well
(Batueva 1987
; Bennett 1966
, 1997
;
Pappas and Bennett 1966
; Rovainen 1979
). A number of the descending axons that were dye coupled to motoneurons originated from segmental descending interneurons that are likely homologous to a class of descending interneurons described in the
goldfish (Fetcho 1992
).
Developmental changes
The most obvious developmental change to occur, the switch from
burst swimming to beat-and-glide swimming, was associated with a
lowering of tail-beat frequency. However, at a cellular level, there
were few changes. The hyperpolarization following fictive swimming
common at day 2 was not observed in day 4 beat-and-glide fictive
swimming. This could be due to the loss of this conductance, to it
being obscured by synaptic activity following the fictive swimming, or
to a lower input resistance of day 4 motoneurons. Rhythmic EPSC
amplitudes increased from day 2 to day 4, which could compensate for
the reduction in input resistance and is likely due to the increase in
size of the unitary AMPA/kainate synaptic events described by
Ali et al. (2000a). There were no significant changes in
most cellular properties including resting membrane potential, fictive
swimming tonic depolarization, action potential amplitude or threshold,
or rhythmic EPSC decay time course.
Comparison with fictive swimming described in other fish
The neural control of swimming has been extensively examined at
the cellular level in the lamprey (reviewed in Grillner et al.
1991, 1998
; Rovainen 1979
) through the use of
the isolated or paralyzed spinal cord preparation. The activity of
motoneurons and unidentified interneurons has been examined during
fictive swimming in dogfish (Mos et al. 1990a
,b
) and
stingray (Williams et al. 1984
) with intracellular
techniques. However, most studies, in fishes other than the lamprey,
have been limited to extracellular recording techniques (goldfish,
Fetcho and Svoboda 1993
; carp, Uematsu et al.
1994
; angelfish, Yoshida et al. 1996
; stingray, Leonard 1986
; dogfish, Roberts 1981
).
During glutamate-induced fictive locomotion in lamprey, phasic
excitation alternates with a phasic inhibition that is mediated by a
glycinergic chloride conductance (Alford and Williams
1989; Dale 1986
; Kahn 1982
;
Russell and Wallen 1983
). These alternating excitatory
and inhibitory oscillations are superimposed on a tonic depolarization
when locomotion is evoked by sensory stimuli (Alford and
Williams 1989
). Fictive swimming is antagonized by
glutamatergic antagonists (Brodin and Grillner 1985
) and
the phasic excitation is mediated by glutamatergic synaptic inputs
having both AMPA and NMDA components (Alford and Sigvardt
1989
; Alford and Williams 1989
; Dale
1986
; Dale and Grillner 1986
; Hagevik and
McClellen 1994
; Moore et al. 1987
).
Spontaneously occurring fictive swimming recorded in stingray
motoneurons (Williams et al. 1984
) is characterized by a
tonic depolarization with superimposed rhythmic PSPs, with little sign
of alternating inhibition. Similarly, fictive swimming recorded from
dogfish motoneurons does not reveal alternating inhibition (Mos
et al. 1990a
).
Blocking glycinergic inhibition, by bath application of strychnine,
increases the rate of fictive swimming in the lamprey (Cohen and
Harris-Warrick 1984; Grillner and Wallen 1980
),
but not in the zebrafish, and synchronizes normal alternating
ipsilateral/contralateral fictive motor output in the lamprey
(Alford and Williams 1989
; Cohen 1987
;
Cohen and Harris-Warrick 1984
). Thus Cohen and
Harris-Warrick (1984)
concluded that the neuronal network
generating the rhythmic excitatory oscillations, observed during
fictive swimming, operates independently of glycinergic inhibitory
connections. The lack of effect of strychnine on the rhythmicity of
zebrafish fictive swimming supports this conclusion. Furthermore,
strychnine does not disrupt the rostral-to-caudal phase lag of motor
output that underlies the propulsion for undulatory swimming in the
lamprey (Alford and Williams 1989
; Cohen
1987
). The present study examined the activities of individual
motoneurons and could not determine whether the rostral-to-caudal phase
lag of motoneuron activity was affected. The ipsilateral/contralateral
alternation during swimming is perturbed by strychnine, resulting in
bilateral contractions resembling the phenotype of accordion
mutants (Granato et al. 1996
).
The synaptic drive to zebrafish myotomal motoneurons is very similar to
that observed in other fishes. The principle difference is the presence
of a tonic glycinergic drive, which we hypothesize to be an adaptation
to the high frequencies of motor output required for undulatory
locomotion in these small larval fish. The patterning of rapid
undulatory movements by the nervous system may underlie fundamental
features in the vertebrate nervous system because this form of
locomotion is observed in phylogenetically distant organisms having a
prevertebrate chordate ancestry (e.g., Lancets; Stokes
1997). Phasic glycinergic inhibition during fictive swimming is
not prominent in the motoneurons of the two elasmobranch fish examined
(Mos et al. 1990a
; Williams et al. 1984
),
suggesting that phasic glycinergic inhibition of motoneurons
is not critical for locomotor activity. The apparent necessity for the
involvement of glycinergic inhibition in ipsilateral/contralateral
alternation likely lies at a premotoneuronal level.
Similarities with mammalian locomotion
The properties of the synaptic drive to zebrafish motoneurons
during fictive swimming have many similarities with the synaptic drive
to feline motoneurons during fictive locomotion. In both preparations,
motoneuron output is determined largely by a rhythmic excitatory
synaptic drive (Jordan 1983; Pratt and Jordan
1987
). In the cat, reciprocal relationships underlying extensor
and flexor motoneuron output during locomotion are believed to be
determined at a premotoneuronal level by interactions among
interneurons forming the flexor reflex afferent pathways
(Jankowska 1992
; Jankowska et al.
1967a
,b
; Schomburg et al. 1998
; Shefchyk
and Jordan 1985a
). Similarly, motoneuron rhythmic activity
remains after glycinergic inhibition is antagonized by strychnine
(Pratt and Jordan 1987
) or nicotinic cholinergic
transmission is antagonized with mecamylamine (Noga et al.
1987
) but not when glutamatergic antagonists are administered
(Douglas et al. 1993
). The synaptic drive to motoneurons is due to a sequential excitatory and inhibitory synaptic drive that
overlap during the step cycle (Orsal et al. 1986
;
Perret and Cabelguen 1980
; Shefchyk and Jordan
1985a
,b
). Two classes of identified interneurons have been
shown to provide this inhibitory synaptic drive to motoneurons during
fictive locomotion. Renshaw cell activity provides an inhibitory drive
concurrent with the excitatory phase of the locomotor drive potential
and Ia inhibitory interneurons provide an inhibitory drive alternating
with the excitatory phase (i.e., midcyle inhibition) of the
locomotor drive potential (Feldman and Orlovsky 1975
;
McCrea et al. 1980
; Noga et al. 1987
;
Pratt and Jordan 1987
). Although both interneurons provide inhibitory drive to motoneurons during fictive locomotion, neither are components of the spinal rhythm generating network (Pratt and Jordan 1987
). It is possible that analogous
(or homologous) inhibitory interneurons, which are not elements of the
spinal rhythm generator, provide the glycinergic drive to zebrafish
motoneurons during fictive swimming. We conclude that there are many
similarities between zebrafish and mammals, in the properties of the
synaptic drive to motoneurons during fictive locomotion, and that the
larval zebrafish is a useful preparation for gaining new insights into the neural control of vertebrate locomotion.
![]() |
ACKNOWLEDGMENTS |
---|
We thank Drs. M. Robertson and J. Dawson for use of the high-speed camera, and G. Pollack and C. Bourque for useful discussions.
This work was funded by the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council. R. R. Buss holds a CIHR Doctoral Research Award.
![]() |
FOOTNOTES |
---|
Address for reprint requests: P. Drapeau, Dept. of Neurology, Montreal General Hospital, 1650 Cedar Ave., Montreal, Quebec H3G 1A4, Canada (E-mail: pierre.drapeau{at}mcgill.ca).
Received 29 December 2000; accepted in final form 26 March 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|