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THE JOURNAL OF COMPARATIVE NEUROLOGY 395:493–509 (1998)
Physiological Properties of the Mauthner
System in the Adult Zebrafish
KOHEI HATTA* AND HENRI KORN
Institut Pasteur, Biologie Cellulaire et Moléculaire du Neurone, INSERM U261,
Département des Biotechnologies, 75015 Paris, France
ABSTRACT
We investigated the morphological and electrophysiological properties of the Mauthner
(M-) cell and its networks in the adult zebrafish (Danio rerio) in comparison with those in the
goldfish (Carassius auratus). The zebrafish M-cell has an axon cap, a high resistivity structure
which surrounds the initial segment of the M-axon, and accounts for an unusual amplification
of the fields generated within and around it. Second, extra- and intracellular recordings were
performed with microelectrodes. The resting potential was ,280 mV with an input resistance
of ,0.42 MV. The M-cell extracellular field was large (10-20 mV), close to the axon hillock, and
the latency of antidromic spikes short (,0.4 milliseconds), confirming a high conduction
velocity in the M-axon. The extrinsic hyperpolarizing potential (EHP), which signals firing of
presynaptic cells and collateral inhibition, was markedly lower at frequencies of spinal
stimulation . ,5/second, suggesting an organization of the recurrent collateral network
similar to that in the goldfish. Inhibitory postsynaptic potentials (IPSPs) were highly
voltage-dependent; their decay time constant was increased by depolarizations. The presynaptic neurons which are numerous could be identified by their passive hyperpolarizing potential
(PHP) produced by the M-spike current. Auditory responses, mediated via mixed synapses
(electrical and chemical), had short delays and hence are well suited to trigger the escape
reaction. The similarities of their properties indicate that the wealth of information generated
over decades in the goldfish can be extrapolated to the zebrafish. J. Comp. Neurol.
395:493–509, 1998. r 1998 Wiley-Liss, Inc.
Indexing terms: axon cap; field effects; electrical inhibition; chemical inhibition; auditory response;
electrical excitation
The zebrafish (Danio rerio), a small teleost, is a useful
vertebrate model for genetic studies of development (Streisinger et al., 1981). It has a relatively short generation
time, produces a large number of eggs, and can be easily
raised in the laboratory. The embryos develop externally
and are transparent such that it is easy to observe their
phenotypes. Genetic screening and analysis are facilitated
by the availability of genetic methods, including those for
generating haploids and gynogenetic diploids and the
viability of frozen sperm. Several laboratories have
screened g-ray- or ethylnitrosourea (ENU)-induced mutants and have reported thousands of mutations in hundreds of gene loci which cause ontogenetic deficiencies in
morphology (e.g., Kimmel et al., 1991; Haffter et al., 1996;
Driever et al., 1996) or in behavior (Westerfield et al., 1990;
Brockerhoff et al., 1995; Granato et al., 1996).
Some of the neurons which develop early (primary
neurons) are identifiable individually (Kimmel and Westerfield, 1990). Examples include the pair of Mauthner (M-)
cells, the largest neurons in the nervous system. They
belong to the set of reticulospinal neurons located in the
r 1998 WILEY-LISS, INC.
hindbrain. The development of their morphology and
sensory inputs has been well described (Kimmel and
Model, 1978; Kimmel et al., 1982; Metcalfe et al., 1986;
Kimmel et al., 1990; Lee and Eaton, 1991), and their
functions in other teleosts and amphibians have been
elucidated. Zebrafish mutants which show abnormal pathfinding of M-axons have been described (Hatta et al., 1991,
1996a; Hatta, 1992; Jiang et al., 1996), and consequently,
the zebrafish can now be used as a model for genetic
analysis of M-cell function. However, in contrast to the
wealth of general information on this neuron’s morphology
and development, there have been few electrophysiological
studies in the zebrafish. It would therefore be useful to
Grant sponsor: INSERM.
*Correspondence to: Dr. Kohei Hatta, Institut Pasteur, Biologie Cellulaire et Moléculaire du Neurone, INSERM U261, Départment des Biotechnolgies, 25 rue du Dr. Roux, 75015 Paris, France. E-mail: [email protected]
Received 30 September 1997; Revised 26 January 1998; Accepted 6
February 1998
494
K. HATTA AND H. KORN
determine whether the zebrafish M-cell and its synaptic
inputs can be analyzed reliably.
The paired M-cells in teleosts and amphibians initiate a
quick escape (or startle) response which removes them
from a dangerous situation and thereby contributes to
survival. M-cells are highly integrative cells for motor
behavior, and a part of the ‘‘brainstem escape network’’
(Eaton et al., 1991). M-cells are unique in the vertebrate
central nervous system because they are readily identifiable both morphologically and physiologically (Faber and
Korn, 1978). In goldfish they display all four physiological
types of synapses: electrical inhibitory, electrical excitatory, chemical inhibitory, and chemical excitatory (Furshpan and Furukawa, 1962; Furukawa and Furshpan, 1963;
Korn et al., 1990). They receive afferent sensory inputs
from a variety of sources, notably auditory, and also many
inhibitory inputs. These inhibitory signals determine the
threshold and the input-output relations of the cell, and
are a major factor in regulation of its function (Faber and
Korn, 1978; Burnod and Korn, 1989; Korn and Faber,
1990). Another important feature is that some of the
inhibitory presynaptic interneurons can be identified electrophysiologically. This facilitates paired recordings from
the pre- and postsynaptic neurons in vivo (Korn and Faber,
1975), often difficult in other vertebrates.
There have been few electrophysiological investigations
of the zebrafish M-cell and all at juvenile stages. The
activity of M-cells in the embryo and larva, monitored
extracellularly, exhibits features that probably reflect the
immaturity of the cell (Eaton and Farley, 1975). For
example, its axon fatigues at relatively low rates (1/second)
of antidromic stimulation. Nevertheless, the transparency
of the embryonic and larval zebrafish brain has enabled
both patch clamp studies of glycinergic currents at synaptic and single channel levels (Legendre and Korn, 1994,
1995), and intracellular Ca21 imaging studies of the reticulospinal neurons during the escape behavior (O’Malley et
al., 1996).
We studied the electrophysiological properties of mature
zebrafish M-cells. Their properties suggest that this preparation will be useful for multidisciplinary studies and for
identifying electrophysiologically the cellular sites affected
by mutations. Preliminary results from this study have
already been published in abstract form (Hatta et al.,
1996b; Korn et al., 1996).
MATERIALS AND METHODS
Adult zebrafish (Danio rerio) were obtained from Charles
River Aquatics (Someren, The Netherlands). Animal care
procedures were in accordance with current requirements
in France.
Physiology
In the course of these experiments, we found that the
adult zebrafish is considerably more fragile than the
goldfish. Thus, it was necessary to modify methods previously used for the studies of other teleosts (Korn and
Faber, 1975; Faber and Korn, 1978) and to minimize the
impact of surgery. Therefore, the lower half of the fish was
first embedded in agarose (4%, w/v) as shown in Figure 1A.
The holding chamber consisted of a Petri dish, two tubes
for perfusion inflow and outflow and a bed of agarose (dark
bottom layer) with an oval depression (not shown) to hold
the fish temporarily. The animal (3-4 cm body length) was
anesthetized in MS 222 (3-aminobenzoic acid ethyl ester,
methanesulfonate salt, Sigma, St. Louis, MO; 0.2 g/l),
placed in the chamber, and an inflow tube, with a mouthpiece made from a pipetteman tip, was placed in its mouth.
A small tissue paper ribbon was inserted into the drainage
tube and wrapped around the opercula. Melted agarose
was cooled until about to solidify, and was poured into the
chamber (gray top layer). Once it had solidified, the ribbon
and the agarose near the opercula were removed to create
a drainage pathway. The animal was continuously perfused through the mouth during the operation with 0.2 g/l
MS 222 in commercially available mineral water (Evian,
BSN-GD, Paris, France) at the rate of ,20 ml/hour. A
ground electrode (chlorided silver wire) was placed next to
the body of the fish.
To stimulate the M-cell from the spinal cord antidromically, bipolar electrodes made of two stainless steel insect
pins, insulated except at their tip with nail polish, were
inserted with a micromanipulator into both sides of the
vertebral column near the posterior end of the dorsal fin.
The satisfactory placement of these electrodes was confirmed by testing for the ‘‘M-reflex’’ in response to electrical
stimulations (typically 2–30 V). This reflex consists of a
generalized contraction of the trunk muscles, accompanied
by a tail flip, a forward flick of the eyes, a rapid protrusion
of mouth, and opercular and pectoral fin movements
(Zottoli, 1978; Faber and Korn, 1978).
A muscular relaxant Pavulon (Pancuronium bromide,
Organon Teknika, Fresnes, France; ,1 µg/g body weight)
was then injected into the body muscles. A hole (around 0.5
mm radius) was cut in the posterior dorsal skull with a
sharp knife and a portion of the cerebellum was exposed
with a fine pair of forceps.
Broken tip glass pipettes for recordings were filled with
3 M KCl or 4 M potassium acetate (KAc); their impedence
was 3-7 MV. The microelecrodes were implanted into the
M-cell at an angle of about 5 degrees posterior from the
vertical. The M-cell soma was typically found at a depth of
0.7–1.2 mm below the cerebellar surface and 100-150 µm
lateral from the midline. The discontinuous single electrode voltage clamp (SEVC) technique (Axoclamp-2A, Axon
instruments, Foster City, CA) was used with sampling
rates of 16 to 23 kHz. To stimulate the M-cell by sound
through the air, sinusoidal waves of different frequencies
and durations were provided by a function generator
(GX240, Metrix S.A., Annecy, France), and their intensities were controlled by an amplifier (Integrated Stereo
Amplifier F245R, Sony, Tokyo, Japan). The loud speaker
was placed to the left and above the fish at a distance of 50
cm. All experiments were performed at room temperature
(18–25°C).
Data were processed by a digitizer (PCM-4/8, Medical
Systems Corp., Greenvale, NY) and stored on tape (VCR:
HR-D158MS, JVC, Tokyo, Japan) for subsequent computer analysis. The programs we used were Trace analyzer
(M. Volaski, Allegheny University of Health Sciences,
Philadelphia, PA) for data collection, superimposition,
averaging and subtraction of individual responses, and
KaleidaGraph 3.0 (Synergy Software, Inc., Reading, PA)
for plotting, curve fitting, and generating histograms.
Morphology
For light microscope observation, adult zebrafish were
anesthetized in MS222 as described above and decapitated
in 0.12 M phosphate-buffered saline (PBS, pH 7.4) on ice.
MAUTHNER SYSTEM IN THE ADULT ZEBRAFISH
495
Fig. 1. A: Top (above) and transverse (below) views of the chamber
adapted to the adult zebrafish for electrophysiological recordings. The
anesthetized animal is laid on a bed of agarose (dark bottom layer), a
perfusion tube is placed into its mouth, and melted agarose (gray top
layer) is then poured into the chamber. A drainage pathway is then
cleared near the opercula for the perfusion flow (indicated by arrows).
Bipolar electrodes made of two stainless steel needles are inserted
near the posterior end of the dorsal fin. A micropipette is inserted
through the cerebellum for recording. The ground electrode is placed
next to the fish body. (See Materials and Methods for details.)
B: Location and size of the Mauthner (M-) cells. Schematic representation of a thick transverse section containing the M-cells with their
major dendrites, axon and axon cap (shaded area). Drawings according to Lee and Eaton (1991) and Wullimann et al. (1996). Scale bar 5
300 µm.
The brain tissues were quickly dissected out, fixed in 1%
glutaraldehyde-1% paraformaldehyde in PBS (Karnovsky,
1967) at 4°C for overnight, and postfixed with osmium
tetroxide (2%) for 2 hours at room temperature. They were
then dehydrated and embedded in Araldite, cut into semithin sections (1-µm-thick) and stained with 1% toluidine
blue (Korn et al., 1973).
determine whether their basic structure, integrative properties, and operations are similar to those in goldfish.
Computer-generated photomicrographs
Photomicrographs were taken on Ilford PanF Plus 50
film. The negatives were printed and scanned at 320 dpi
resolution on a Horizon scanner (AGFA, Paris, France)
controlled by a Power Macintosh 9500/180 using a Photoshop plug-in module (Fotolook, AGFA, Paris, France).
Adobe Photoshop 4.0 (Adobe Systems, Inc., Mountain
View, CA) was used to assemble the figures with a final
resolution of 320 dpi. Linear contrast and brightness
adjustments were made to the scanned image until the
gray range and contrast of the electronic version most
closely approximated that of the original photograph. The
final figures were obtained with a Pictography 3000 photographic printer (Fuji, Bois d’Arcy, France).
RESULTS
The morphological and electrophysiological characteristics of the zebrafish M-cell network were studied to
Morphology of the axon cap
The position and overall morphology of the M-cells in the
adult zebrafish brain have been described in a horseradish
peroxidase (HRP) backfill study by Lee and Eaton (1991;
Fig. 1B). The M-cells are bilateral, located in the medulla
under the cerebellum (C) and behind the optic tectum (OT)
near the point of entry of the VIIIth nerve. They have two
principal dendrites, one lateral and one ventral. As in
other teleosts (Zottoli, 1978) their axon crosses the midline, turns caudally, and extends through the contralateral
side of the spinal cord toward the tail. The unmyelinated
initial segment of the M-axon is surrounded by a specialized neuropil called the axon cap (AC; Bartelmez, 1915).
We first analyzed the morphological features of the AC.
A light micrograph of a semithin plastic section stained
with toluidine blue shows the left M-cell soma (Ms) with its
AC located dorsomedially and a middle portion of the right
M-cell’s lateral dendrite (Fig. 2A). The adjacent sections at
larger magnification (Fig. 2B1-3) reveal the typical structure of an AC which shares several common characteristics
with that of goldfish. They are: (1) its outer border is
covered with a wall-like epithelial structure consisting of
layered glial cells called cap cells and designated as the cap
496
wall (Robertson et al., 1963; Nakajima and Kohno, 1978;
Fig. 2B1); (2) it contains a dense neuropil comprised of
numerous afferent fibers; (3) axons inside the AC lack
myelin. Three types of axons were distinguished: (a) the
M-axon runs dorsomedially and a myelinated portion (Fig.
2B1), an unmyelinated initial segment (Fig. 2B2), and an
axon hillock (Fig. 2B3) were observed; (b) smaller myelinated afferent fibers (Fig. 2B1) probably part of the spiral
axons (Bodian, 1937); and (c) large myelinated fibers (Fig.
2B1), presumably axons of inhibitory interneurons or
‘‘PHP (passive hyperpolarizing potential)-cells’’ (Faber and
Korn, 1973; Korn and Faber, 1975), were also present. As
in the goldfish (Korn et al., 1978), these axons dilate to
form large bulbs or swellings at the border of the AC
(Fig. 2B2).
There are some differences between the ACs of the
zebrafish and goldfish. In the goldfish, the neuropil can be
resolved into two distinct regions: a central core portion
which contains the spiral fibers, and a peripheral region
which contains most inhibitory axons (Nakajima and
Kohno, 1978). The central core of the AC stains more
heavily than the peripheral part (Bartelmez, 1915). In
contrast, in the adult zebrafish, the neuropil was relatively
uniformly stained (Fig. 2B1-3), as already described in the
larva (Eaton et al., 1977a; Kimmel et al., 1979). More
major differences between other teleosts have been reported (Otsuka, 1964; Zottoli, 1978; Kimmel and Model,
1978). Generally the basic organization of the M-cell
structure is very similar in the two species. The diameter
of the AC is ,60 µm in the zebrafish assuming 5% to 10%
shrinkage during histological preparation and ,100 µm in
the goldfish (Nakajima, 1974). This is a small discrepancy
given the 3- to 5-fold difference in body lengths.
Generation of field effects and of chemical
inhibition in the goldfish, a brief summary
We found that as in the goldfish, the M-cell collateral
inhibitory network of the zebrafish produces both electrical and chemical inhibitions mediated by the same set of
interneurons. Before describing the results leading to this
conclusion, we will summarize the major features of the
M-cell inhibitory networks in goldfish and the various
electrical events that they generate.
Antidromic stimulation of the M-axon in the spinal cord
activates the recurrent network (Fig. 3A). This results in a
disynaptic excitation of PHP-cells which establish chemical inhibitory synapses on the M-cell, and paired electrophysiological recordings can be made intracellularly from
the identified PHP-cells and the M-soma, or extracellularly. The sequences of responses evoked by this procedure
are schematically represented in Figure 3B.
Passive hyperpolarizing potentials in the interneurons. The network inhibitory interneurons are called
PHP-cells because when the M-cell is excited they exhibit a
passive hyperpolarizing potential or PHP, the latency and
duration of which coincide with the M-spike (B1 versus
B2-B3). This potential is generated by the M-spike action
currents (Fig. 3C1). Figure 3C illustrates the intra- and
extracellular distribution of current. As the extracellular
resistance in the AC is relatively high, some current
appears to be channeled through the parallel resistive
path provided by the interneurons (iPHP). This PHP has
an inhibitory effect on the interneurons because it hyperpolarizes their excitable region. It can also be followed by a
postanodal rebound excitation, which has a role in synchro-
K. HATTA AND H. KORN
nizing the discharge of neighboring PHP-cells (Faber and
Korn, 1983).
Extrinsic hyperpolarizing potentials. In the axon
cap the large negative M-spike field is followed by a series
of positive peaks named the extrinsic hyperpolarizing
potential, or EHP (Furukawa and Furshpan, 1963; Fig.
3B2). The latency and components of the EHP coincide
with the action potentials in PHP-cells (B2 versus B1). Its
size and electrical inhibitory effect on the M-cell are
consequences of the high resistance of the AC (Fig. 3C2).
Note that although PHPs are produced by the action of one
neuron (i.e., the M-cell), at least 40-50 synchronously
active cells contribute to the EHP (Korn and Faber, 1975).
Chemical inhibition. Cl2-dependent inhibitory postsynaptic potentials (IPSPs) recorded in the goldfish M-cell
are glycinergic (Furukawa et al., 1964; Faber and Korn,
1982; Korn et al., 1990) and appear as depolarizations
when the neuron is chloride-loaded (Fig. 3B3). In goldfish,
it has been directly shown (Korn and Faber, 1975, 1976)
that chemical and electrical inhibitions are produced by
the same set of interneurons. This is strongly supported by
the observation of the presence of a PHP in interneurons
near the M-cell and the fact that the latency of the IPSP
following spinal stimulations is compatible with three
synaptic delays.
Localization of the M-cell in the zebrafish
As in other species (Furukawa and Furshpan, 1963;
Faber and Korn, 1978), extracellular fields helped localize
the M-cell in the zebrafish. Multiple tracks were used to
maximize the size of the M-spike and of the EHP. The
former had a latency of about 0.2–0.7 milliseconds and its
maximal amplitude was 210 to 220 mV at the center of
the AC (Fig. 4B, bottom; also see Fig. 6A). Within this
region, this negative component was followed by an EHP,
which increased in magnitude as the electrode approached
the ‘‘spike focus,’’ i.e., the center of the AC. Furthermore,
when the electrode was moved laterally along the M-cell
lateral dendrite, the field became biphasic, positivenegative, a waveform indicative of passive spike propagation along the dendrite (Furshpan and Furukawa, 1962).
The amplitude of the extracellular M-spike was 210 to
220 mV. That of the corresponding action potential recorded intracellularly in the soma was ,20 to 30 mV. Thus,
the full size of the transmembrane potential change can
only be determined by calculating the difference between
Fig. 2. Morphological features of the zebrafish M-cells and of their
axon cap. Light micrographs of semithin (1 µm) plastic sections
stained with toluidine blue. Transverse sections, dorsal, up. A: Photograph at low magnification showing the location of the left M-soma
(Ms) and axon cap (AC), and the right lateral dendrite (LD). An arrow
indicates the midline. B1–B3: Adjacent sections at larger magnification. Midline to the right. B1: The axon cap contains numerous small
axons and glial processes, and densely packed of cytoplasmic membranes. Its outer border is covered by a wall-like structure consisting
of a layer of glial cells (G; classified as astrocytes) or cap cells. Note the
M-axon (M-ax), and the presence of several small myelinated fibers
(crossed arrow) probably spiral fibers running in parallel with it. They
lose their myelin sheath where they enter the axon cap. Large
myelinated fibers, presumably axons of passive hyperpolarizing potential (PHP) inhibitory cells, also lose their myelin sheaths at their
boundary (arrow). B2–B3: Initial segment (IS) and axon hillock (AH)
of the M-axon. At this level, most of the large fibers dilate and form
large spherical or ovoid bulbs (asterisks). Scale bars 5 50 µm in A,
10 µm in B1–B3.
MAUTHNER SYSTEM IN THE ADULT ZEBRAFISH
497
Figure 2
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K. HATTA AND H. KORN
Fig. 3. Correlation of electrical events recorded pre- and postsynaptically with the anatomical characteristics of the M-cell system in the
goldfish. A: Recurrent inhibitory network. When the M-axon is
stimulated antidromically, it activates a class of intermediate or
cranial relay cells (CR). These cells excite a group of inhibitory
interneurons (passive hyperpolarizing potential [PHP]-cells), the axons
of which enter the axon cap (shaded area) and synapse on the M-cell.
Arrows indicate the direction of impulse propagation. B1–B3: Consequences of M-cell activation. Scheme of the events recorded intracellularly from a PHP-cell (B1), extracellularly in the axon cap (B2), and
intracellularly from a Cl2-loaded M-cell (B3). The field effects (shadowed) and spikes which generate the action currents providing them,
are linked by dashed lines; note that the latencies of the PHP and of
the M-cell spike recorded extracellularly (B2) or intracellularly (B3)
are identical. Subsequently, the action potentials in the interneuron
(B1) and the components of the extrinsic hyperpolarizing potential
(EHP; B2) coincide with each other. The inhibitory postsynaptic
potential (IPSP) produced by chemical transmission from the PHPcells is delayed (B3) because it occurs after three synaptic delays.
C1–C2: Equivalent circuits accounting for the generation of the PHP
(C1) and of the EHP (C2); intracellular channeling of action currents
due to the high extracellular resistance (Re) results in reciprocal
hyperpolarizing effects. Two adjacent neurons are shown in each
diagram. They are the M-cell, with its soma and axon, and a PHP-cell
(Int), that sends its axon towards M-cell’s axon hillock. These two
units are coupled through Re. C1: The M-cell is active (battery) and it
produces an action potential at the initial segment and axon hillock;
action currents (arrows) leaving the cell at its soma return to the spike
focus or pass through both Re and the interneuron (iPHP), hyperpolarizing the latter at the point of entry. C2: The interneuron is active
(battery at its soma). Resultant currents flowing extracellularly to the
interneurons return to the soma along either Re, or, more importantly,
along the parallel resistive pathway provided by the M-cell (iEHP).
This current produces a positive extracellular field near the initial
segment and axon hillock of the M-cell, and causes hyperpolarization
at this level. Note that both field effects are inhibitory because
hyperpolarizations are imposed on the spike-generating membranes;
in contrast, the channeled currents exit the affected cell across
electrically inexcitable regions. (C1, C2 are modified from Faber and
Korn, 1989.)
the intracellular (Vm) and extracellular (Vout) potentials
(Figs. 3B2-B3 and 4B). The true spike amplitude was thus
calculated to be ,40 to 50 mV. In the axon, however, it was
as high as 70 to 80 mV, and in the lateral dendrite, it
decreased with distance from the soma to 10 mV or less.
The M-axon conduction velocity in goldfish varies as a
function of body length with a maximum of about
100 m/second for a 9.5 cm fish (Funch et al., 1981). This is
about twice the value found in the adult zebrafish. The
similarity of the conduction time in both species, i.e.,
0.2–0.7 milliseconds versus 0.3-1.2 milliseconds in the
goldfish (Furshpan and Furukawa, 1962), is consistent
with the notion that the escape behavior has similar
kinematics (Eaton et al., 1977b) in the two species.
Axonal properties, conduction
velocity of the M-axon
As the M-axon is the largest fiber in the spinal cord
(Furshpan and Furukawa, 1962), it presumably has the
fastest conduction velocity. The conduction velocity was
measured (Fig. 4A), from both intracellular and extracellular recordings (Fig. 4B). Both methods yielded the same
results: an average conduction velocity of 45 m/second 6
9.6 (S.D., n 5 28; Fig. 4C).
Membrane properties
The resting potential of the recorded cells was in the
range of 70 to 80 mV, similar to that in the goldfish. The
M-cell’s instantaneous current-voltage relation obtained
in the soma with the voltage clamp technique using a
MAUTHNER SYSTEM IN THE ADULT ZEBRAFISH
499
Fig. 4. Conduction velocity of the zebrafish M-axon. A: Diagram of
the experimental set-up for antidromic stimulation (Stim) of the
M-cell at the level of the caudal spinal cord and for recording (Rec) at
the level of the M-soma or initial segment. d is the distance between
the sites of recording and of stimulation (calculated from nine experi-
ments). B: Antidromic M-spikes were recorded intracellularly (upper
trace) or extracellularly in the AC (lower trace). Note that the
measured latencies (t), indicated by the dashed lines, were identical.
C: Histogram of the M-spike’s latency measured extracellularly (open)
and intracellularly (shadowed), in 28 experiments.
KAc-filled microelectrode was linear (Fig. 5A1-2, open
circles). The mean input resistance calculated from the
slope of this relationship for 17 cells was 0.42 MV 6 0.20
(Fig. 5B), which is about twice as large as that of the
goldfish (Fukami et al., 1965; Faber and Korn, 1986). This
difference is consistent with the difference in size (Faber
and Korn, 1988; Edwards et al., 1990).
There was a marked voltage- and time-dependent change
in the leakage currents (Fig. 5A1). During the larger
hyperpolarization, there was a pronounced rapid relaxation of the inward currents. When the cell was hyperpolarized by more than 20 mV, the current remaining after 15
milliseconds was less than 50% of the peak current. This
confirms that the major component of the M-cell’s input
conductance is voltage-dependent, as it is inactivated by
hyperpolarization (Faber and Korn, 1986). Thus, the M-cell
has a high resting K1 conductance, at least 50% of which
can be readily inactivated by brief hyperpolarizations. In
contrast, the low conductance for outward rectification
present in the dendrites of the goldfish was not observed in
zebrafish because recordings were somatic. Instead, there
was a slight increase in the outward current during
depolarizations, which may reflect activation of voltagesensitive K1 channels at the axon hillock and initial
segment.
Identification and properties
of collateral inhibition
External: Extrinsic hyperpolarizing potential (EHP).
The EHP in the AC signals firing of collateral PHP neurons
as explained above. Its waveform was similar to that in the
goldfish in some cases, containing 2-4 components (n 5 6 of
16 recordings). Figure 6A illustrates a case in which it had
three peaks and was abolished when the frequency of the
spinal stimulation was increased from 5 to 10/second. Most
often, however, it lasted longer and exhibited as many as
12–27 components (n 5 10 of 16). Figure 6B1 is representative of this situation where the number of the peaks
fluctuated from 3 to 16 with a total duration of 2.7 to 14
milliseconds. The mean interval between the successive
wavelets was 0.9 milliseconds, suggesting a frequency of
about 1.1 kHz for presynaptic firing. In two cases the
number of the components increased as the strength of the
spinal stimulus was raised beyond the threshold, suggesting recruitment of spinal or other afferent inputs to the
M-cell circuits (See Discussion). Conversely, an EHP was
observed in some cases even when the stimuli failed to
excite the M-axon of the recorded cell (Fig. 6B2,B3), which
is to be expected, assuming activation of the contralateral
M-axon with collaterals of networks converging onto com-
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K. HATTA AND H. KORN
Fig. 5. Current-voltage relations of the M-cell, obtained in one
experiment with the single electrode voltage clamp (SEVC) technique,
demonstrating hyperpolarizing inactivation of the instantaneous leakage current. A1–A2: Voltage clamp currents and corresponding currentvoltage plot, respectively. The soma was voltage-clamped at a chopping rate of 18 kHz with a 4 M KAc-filled electrode. A1: Family of
currents (above) evoked by 15 millisecond command pulses (below) in
the depolarizing (15, 110, 115, 120 mV) and hyperpolarizing (210,
215, 220, 225, 230, 235 mV) directions. Note that with the larger
hyperpolarizations, there was a rapid decline in the inward currents
from their initial peaks, and that there was a slow increase in the
outward current with larger depolarizations, presumably due to the
onset of delayed rectification at the axon hillock. A2: Plots of the
voltage clamp currents (ordinate, I) as a function of membrane
potential (abscissa, V). The peak currents (open circles) were linearly
related to membrane potential (input conductance 5 3.38 µS) whereas
the currents measured after 15 milliseconds (filled circles) did not
increase in direct proportion to the voltage steps (corresponding
symbols in A1 designate times at which the currents were measured).
B: Histogram of the input resistance of the M-cell in 17 experiments.
The leakage conductances used for calculation were measured at the
beginning of the command pulses.
mon presynaptic interneurons (Furukawa and Furshpan,
1963).
Intracellular recordings: Presynaptic interneurons
(PHP-cells). Presynaptic interneurons were identified
by the presence of a PHP (Fig. 7A1,B1). The latency of the
PHP coincided with that of the M-spike (Fig. 7A2 versus
A3). The net value of this transmembrane potential (DV in
A3,B2) was determined by comparing the intracellular
potential with the corresponding extracellular potential.
Its amplitude varied from 2 to 5 mV (3.3 mV 6 1.2, n 5 6)
and was insensitive to the quality of the penetration. The
PHP was often followed by an excitatory postsynaptic
potential (EPSP; Fig. 7A3) the latency of which was 0.45
milliseconds 6 0.15 (n 5 7), relative to the peak of the PHP.
This lag presumably reflects in part the synaptic delays
between the M-collaterals and the cranial relay cells (CR)
and between the CR-cells and the recorded neurons (see
Discussion). As expected, these EPSPs could trigger action
potentials. It should be noted, however, that although
some PHP-cells were only briefly excited (2–3 spikes
maximum, in 3 of 6 cells) as in the goldfish (Korn and
Faber, 1975), others fired repetitively with 23 to 27 spikes,
as expected from the shape of the EHP. The fact that the
frequency of the presynaptic spikes (Fig. 7B1) coincides
with that observed extracellularly for a population of
interneurons indicates that this property of zebrafish
inhibitory cells is not an artifact.
Fatigability of the collateral network. The EHP is
diminished and eventually abolished at relatively low
stimulation frequencies in the goldfish (1–5/second; Furshpan and Furukawa, 1962), because M-cell axon synapses
onto CR fatigue (see Fig. 3A for network diagram and
Hackett and Faber, 1983; Faber et al., 1989). This was also
the case in the zebrafish (Fig. 8A), although higher frequencies (no less than 10/second) were required to totally
eliminate both the EHP and the corresponding IPSPs (Fig.
8B). Figure 8A and B shows that this decrease was gradual
with both EHP and IPSPs becoming progressively smaller
as the frequency of the stimulation increased. The similar
frequency dependence of the electrical and chemical inhibitions is consistent with the notion that they are mostly
generated by the same populations of presynaptic neurons
(Korn and Faber, 1976).
In intracellular recordings at high frequency (5 and 10/s
in Figure 8B), a small depolarization often persisted, albeit
with a delayed onset. This was probably a component of
the IPSP generated in the lateral dendrite by another set
of inhibitory cells (Furukawa et al., 1964; Faber and Korn,
1978).
Chemical inhibition
The kinetics of the chemical inhibition induced in the
M-cell by the collateral network was studied by three
methods. All gave similar results.
First, the inhibitory conductance underlying the IPSP
was revealed: it produced a reduction, or shunt, of the test
antidromic spike with paired spinal stimulations, delivered at progressively increasing intervals, using KAc-filled
electrodes. Figure 9A shows superimposed traces from one
such experiment, the shunt being maximal at the shortest
intervals, and gradually diminishing, to cease at around
20 to 30 milliseconds following the first stimulus.
MAUTHNER SYSTEM IN THE ADULT ZEBRAFISH
501
Fig. 6. Evidence that collaterals of both M-axons activate common
inhibitory interneurons. A: Superimposed extracellular recordings
obtained in the AC after spinal cord stimuli above threshold for firing
the M-cell. The extrinsic hyperpolarizing potential (EHP) disappeared
when the frequency of antidromic stimulation was raised from 5 to
10/second. B1–B3: Superimposed extracellular responses induced by
stimuli at threshold for firing the M-cell at one-third second recorded
in the AC of a different fish. B1: The antidromically evoked M-spike
was followed by an EHP (arrow) or by a prolonged oscillatory field
potential which may have been produced by orthodromic activation of
spinal afferents. B2: The stimuli failed to excite the recorded M-cell
but there remained an EHP resulting, presumably, from activation of
the opposite M-axon. B3: Comparison of the EHPs with and without a
preceding M-spike. Their identical waveform can be explained by the
fact that collaterals of both M-axons and/or spinal inputs converge
onto common interneurons.
Second, with KCl-filled electrodes, the Cl2-inverted collateral IPSP was a frank depolarization (Fig. 9B), of as
much as 20–40 mV.
Third, the underlying inhibitory postsynaptic currents
(IPSCs) were monitored by voltage-clamp at resting potential (Fig. 9C).
The kinetics of the conductance change were the same
with all three methods. The total duration was about 30
milliseconds, and the decay time course was relatively
simple, suggesting that in the case of repetitive firing, the
last spikes in presynaptic trains does not contribute
significantly to the inhibition.
Voltage dependency of Cl2 currents. M-cell’s Cl2
channels in the adult goldfish are activated by glycine and
are strongly voltage-dependent (Faber and Korn, 1987).
This may also the case in the adult zebrafish (Fig. 10).
Specifically, the exponential decays of the collateral IPSCs
were highly voltage-sensitive (Fig. 10A1-A2) in both Cl2loaded and nonloaded cells (Fig. 10B). The peak synaptic
conductance was lower at stronger hyperpolarizations
(e.g., at 240 mV in Fig. 10A2). However, the major effect
was on the IPSC decay time constant tD, which increased
logarithmically as a function of membrane potential, so
that the IPSC was significantly prolonged with depolarizations. As postulated for goldfish, this may provide a
mechanism for enhancing inhibitory responses in the face
of increased excitations (Faber and Korn, 1987).
Excitatory inputs: Responses to sounds
The properties of the excitatory postsynaptic potentials
(EPSPs) evoked in the M-cell by sounds of different
intensities and tones were examined using KAc-filled
electrodes. The top diagram in Figure 11 illustrates the
afferent auditory network in the goldfish. Figure 11A1-B2
shows potentials evoked by tone bursts and recorded in the
M-soma of the zebrafish. EPSPs were induced by sounds
(500 Hz) of various intensity, the largest of which could fire
the cell. The duration of the initial component of the EPSP
(crossed arrow) was short (about 1 millisecond), suggesting the involvement of gap junctions. Its amplitude increased with sound intensity. It was followed by longer
lasting components that could be mixed, i.e., chemical and
electrotonic as, for example, in the lateral vestibular
nucleus of the toadfish (Korn et al., 1977). A comparison of
the traces superimposed in Figure 11A2 demonstrates that
the latency of the auditory response is variable, decreasing
502
K. HATTA AND H. KORN
Fig. 7. Passive hyperpolarizing potential (PHPs) evoked in presynaptic interneurons by M-cell antidromic spikes. Inset: Diagram of the
experimental set-up. A microelectrode was inserted into a M-cell
presynaptic interneuron and responses were evoked in the M-cell by
spinal cord stimulation (Stim). A1: Superimposed intra- and extracellular recordings obtained with spinal stimuli that were above the
threshold for M-cell activation. The stimulation evoked a hyperpolarization, or PHP, followed by a prolonged depolarization. A2: Recording
from the axon cap during the same experiment. A3: Net transmem-
brane hyperpolarization calculated as the difference (DV 5 22.5 mV)
between the intra- and extracellular potentials illustrated in A1. Note
that the latencies of the M-spike in the axon cap and in the PHP were
identical (dashed line). B1: Recordings from another PHP-neuron and
same presentation as above. The PHP was presumably followed by a
postanodal rebound and a recurrent excitatory postsynaptic potential
(EPSP) resulting in a high frequency repetitive firing of the cell.
B2: Net hyperpolarization during the PHP (DV 5 22.0 mV) calculated
as for A3. The black dot indicates a PHP in all records.
with increasing sound stimulus: for sounds of 50, 60, and
70 dB, the latency was 3.8 6 0.3, 2.8 6 0.4, and 2.5 6 0.2
milliseconds, respectively (n 5 4). A similar relation has
also been reported for the goldfish afferent eighth nerve
fibers (Furukawa, 1981). Responses to tones of 500 Hz and
1 kHz were compared. Their respective latencies and time
courses (50 and 70 dB) were the same (Fig. 11B1-B2).
weaker than the goldfish, an impediment for neurophysiological studies. Nevertheless, with some caution, this in
vivo preparation appears to be well suited for a variety of
studies. For example, it may allow identification of the
sites (network or synapses) affected by genetic mutations
causing defective locomotion and behavior.
DISCUSSION
Our data for the zebrafish M-system are very similar to
those reported for the goldfish. The few differences between the two systems are mostly quantitative, despite the
fact that these two species belong to distinct subfamilies of
Cyprinidae with different systematics and biogeography
(Chen et al., 1984; Wu, 1987; Nelson, 1994). Thus, we
conclude that the basic properties of the M-network and
afferent synapses onto the M-cell are conserved in these
two teleosts, and that the detailed knowledge which has
been accumulated on the goldfish M-system and its afferent inputs (Korn et al., 1990) is valid for the zebrafish.
Overall, the zebrafish preparation provides a useful genetic model. However, it is considerably smaller and
Properties of the axon cap
The structure of the AC is more uniform in the zebrafish
than in the goldfish, possibly because its central core is
larger and its peripheral part is less prominent. On the
other hand, it has been estimated that the goldfish AC has
at least a 2- to 2.5-fold greater resistivity than the surrounding tissues (Korn and Faber, 1975). This high extracellular
resistivity can be attributed to the dense packing of the
fine neuronal processes, to a restricted extracellular space
(Nakajima and Kohno, 1978), and to the specialized glial
barrier formed by the cap cells. It contributes to the large
size of the extracellular spike recorded in the axon cap and
the reciprocal electrical inhibitions between the M-cell and
its presynaptic inhibitory interneurons (Furshpan and
Furukawa, 1962; Korn and Faber, 1975) via characteristic
field effects (Faber and Korn, 1989). We showed here that
MAUTHNER SYSTEM IN THE ADULT ZEBRAFISH
Fig. 8. Frequency dependence of collateral inhibition. Superimposed traces recorded extracellularly in the axon cap (A) and intracellularly from the M-cell soma of a different fish, with KCl-filled
electrodes (B). The responses were evoked antidromically by spinal
stimuli, which were suprathreshold for the M-axon. Note that both the
503
extrinsic hyperpolarizing potential (EHP) in A and the inhibitory
postsynaptic potential (IPSPs) in B became progressively smaller and
more delayed as the stimulus frequency was increased, and that they
almost completely disappeared at 10/second (see text). The crossed
arrow indicates a late dendritic IPSP.
Fig. 9. Time course of the M-cell collateral inhibition obtained from
three different experiments. A: Multiple exposures of responses produced in a M-cell by paired spinal stimulations separated by progressively longer intervals, and recorded with a KAc-filled microelectrode.
Note the reduced amplitude of the test spikes, due to the increased
membrane conductance. B,C: Time course of the depolarizing postsynaptic potential assessed with a KCl-filled microelectrode (B) and of the
inhibitory postsynaptic current monitored in the single electrode
voltage clamp (SEVC) mode (chopping frequency, 18 kHz) by using a
KAc-filled electrode, both inserted into the M-cell soma (resting
potential, 280 mV; C). Note that the kinetics of the shunt, of the IPSP
and of the inhibitory postsynaptic currents (IPSC) almost coincide
with each other.
MAUTHNER SYSTEM IN THE ADULT ZEBRAFISH
505
Fig. 10. Voltage dependence of the inhibitory synaptic currents.
A12A2: Collateral inhibitory postsynaptic currents (IPSCs) recorded
in the single electrode voltage clamp (SEVC) mode using a KAc-filled
microelectrode. A1: Superimposed currents recorded in the soma at
the indicated potentials relative to the resting potential (RP) with and
without antidromic stimulations. A2: Family of the net collateral
synaptic currents determined by subtraction of the leak currents in
A1. Note that the IPSC decays more rapidly as the cell is more
hyperpolarized, and that the magnitude of the inward current does not
increase in proportion to the level of hyperpolarization. B: Evidence
that a single steep exponential relates tD to membrane potential,
regardless of the internal Cl2 concentration. Semilogarithmic plot of
the time constant (tD) of IPSC decay (ordinate) against the membrane
potential (Vm, abscissa); data points were obtained under different
experimental conditions, i.e.; IPSCs evoked in the presence (two
experiments, filled symbols) and in the absence (four experiments,
clear triangles, clear and quartered squares and crosses, respectively)
of Cl2 loading. All recordings were from the M-cell soma, except for
one, which was obtained from the proximal part of the lateral dendrite
(filled triangles). The straight line fitting all data points was derived
according to the expression tD 5 t0 exp(Vm/B); (n 5 22, r2 5 0.77, P ,
0.01).
all the anatomical features accounting for the high resistivity of the AC can be found in the zebrafish by light
microscopy. As a consequence, we observed the typical
electrical ‘‘signature’’ of M-cell activation, including its
large extracellular spike followed by an EHP, and PHPs
were also recorded in presynaptic neurons.
more often repetitive than in the goldfish. The EHP was
also prolonged when recorded extracellularly and thus the
sustained firing of PHP-cells could not have been due to
damage during intracellular recordings. In other cases,
the number of peaks in the EHP and its duration were
found to fluctuate, and/or to increase when the intensity of
the spinal stimulations was raised. Such additional EHPs
evoked by suprathreshold stimuli were obviously distinct
in their origin from those induced by activation of the
collateral network. They were due to the recruitment of
additional afferents having higher thresholds than that of
the M-axon, which has the lowest threshold of any axon at
the spinal level (Furukawa and Furshpan, 1963).
Chemical inhibition of the M-cell is chloride-dependent
and lasts for about 20 to 30 milliseconds (t 5 ,8 milliseconds, RP 5 280 mV); it is largely glycinergic (and therefore strychnine-sensitive) in both goldfish and zebrafish
(Furukawa et al., 1964; Faber and Korn, 1982; Korn et al.,
1990; Legendre and Korn, 1994; this work, not shown).
Furthermore, collateral IPSCs are highly voltage-dependent, with their decay time constant increased by depolarizations. A similar voltage dependency has been reported
Similarities and differences in the M-system
The design and flexibility of the recurrent networks are
also remarkably similar in both species. For example,
EHPs were detected even in the absence of an M-spike,
suggesting that the collateral inhibition feeds back on each
M-cell via the bilateral projection of CR neurons (Hackett
and Faber, 1983; Faber et al., 1989). However, the collateral network fatigues at higher frequencies of antidromic
stimulation in the zebrafish (responses disappear at 10/
second versus 5/second in goldfish), due presumably to the
properties of the synapses between the M-cell’s axon
collaterals and CR neurons (Hackett and Faber, 1983).
Thus, both chemical and electrical inhibitions are more
powerful in the zebrafish. In agreement with this notion,
the EHP could last longer, and PHP-neuron firing was
506
K. HATTA AND H. KORN
Fig. 11. Sound-evoked excitatory responses of the M-cell.
Top: Diagram of the afferent auditory network. VIIIth nerve primary
fibers (VIII n.) activated by the hair cells terminate on the lateral
dendrite of the ipsilateral M-cell with mixed electrical (el.) and
chemical (ch.) synapses. They also excite a class of commissural
inhibitory interneurons (Comm. inhib.) which are presynaptic to both
the ipsi- and the contralateral (not shown) M-cells. A1–B2: Potentials
evoked by tone bursts (top traces, except for A2) of indicated intensities and recorded in the M-soma with KAc-filled electrodes (single or
superimposed traces). A1: Excitatory postsynapatic potentials (EP-
SPs) evoked by eleven cycles of 500 Hz. The higher intensity sound
fired the cell (arrow). Note that the initial component of the excitatory
potential (crossed arrows) is of short duration (about 1 millisecond),
and is followed by long lasting components, suggesting transmission
via electrical and chemical synapses, respectively. A2: Superimposition of the traces illustrated in A1 but shown at a faster sweep speed.
Note that the onset of the synaptic potential (arrows) becomes quicker
as the sound stimulus is stronger. B: Responses to tones of different
frequencies (B1: 500 Hz, B2: 1 kHz) and of moderate intensities (50
and 70 dB). The M-cell reacted similarly to sounds of either tone.
in the adult goldfish (Faber and Korn, 1987), and for the
single channels in embryonic zebrafish (Legendre and
Korn, 1995). Functionally, the increase in tD during depo-
larization may provide an important inhibitory safety
factor that would compensate for any reequilibration of
Cl2 and diminution of the IPSP driving force.
MAUTHNER SYSTEM IN THE ADULT ZEBRAFISH
The lateral dendrites also share common features. When
the electrode was moved posteriolaterally from the M-cell
soma and placed in the vicinity of this process, the
antidromic extracellular field became positive-negative, as
described for the goldfish (Furshpan and Furukawa, 1962).
It was smaller and monophasic in more distal regions (not
shown). This suggests that spikes generated at the initial
segment of the M-axon propagate passively in the lateral
dendrite. Intradendritically, we recorded additional components of the collateral IPSPs, with a latency ,1 to 1.5
milliseconds longer than that in the soma (Fig. 8B), which
correspond to dendritic collateral IPSPs in goldfish (Furukawa et al., 1964; Faber and Korn, 1978). Furthermore,
the responses to sound bursts contained both fast electrical and slow, presumably chemical, components, suggesting mixed transmission from primary VIIIth nerve afferents as in goldfish (Furshpan, 1964; Nakajima and Kohno,
1978; Lin and Faber, 1988).
Zebrafish M-system as a model for studying
motor control and neuronal diseases
Functionally, the M-cell is a major integrating neuron
that triggers the escape command to motor networks
(Eaton and Bombardieri, 1978; Eaton et al., 1991; Korn
and Faber, 1996). It is also involved in a variety of related
motor behaviors in different contexts, such as hatching
(Eaton et al., 1991) and prey capture (Canfield and Rose,
1993). Since the discovery of its large axon in the cord
(Mauthner, 1859), the M-cell has been a valuable vertebrate model in diverse neurobiological disciplines (Piatt,
1950; Stefanelli, 1951). The M-cell’s afferent inputs also
exhibit a strong history-dependent plasticity, including a
long-term potentiation of the inhibitory synapses (Korn et
al., 1992; Oda et al., 1995) and of both the electrotonic and
chemical components of the excitatory VIIIth nerve junctions (Yang et al., 1990; Pereda and Faber, 1996).
Inhibition in neural networks is one of the major mechanisms regulating motor behavior. In humans, point mutations within the glycine receptor a1 subunit gene have
been found to underlie hypertonic motor disorders (hyperekplexia: startle disease such as ‘‘stiff baby syndrome’’).
The substitution of arginine 271 with leucine or glutamine
(Shiang et al., 1993) drastically reduces agonist-binding,
glycine-induced currents and elementary chloride conductance (Langosch et al., 1994; Rajendra et al., 1994).
Mutant mice with similar phenotypes, spasmodic (spd)
and oscillator (spdot), carry recessive mutations of the a1
subunit gene (Glra 1): the substitution of the Alanine 52
lowering the agonist affinity, and a microdeletion resulting
in a functional null allele, respectively (Ryan et al., 1994;
Saul et al., 1994; Buckwalter et al., 1994; reviewed in
Becker, 1995). Mutant zebrafish with motor defects have
already been isolated (Granato et al., 1996), and cDNA
encoding the a1 subunit of the zebrafish glycine receptor
has been recently cloned (David-Watine et al., 1997) and
expressed in oocytes (Bregestovski et al., 1997). Hence, the
zebrafish M-system offers a particularly suitable model for
studying the consequences of such mutations at the synaptic, cellular network, and behavioral levels. Results obtained in zebrafish models by combining electrophysiological studies with molecular tools may contribute
significantly to our understanding of genetically acquired
motor disorders, including those described in mammals
and primates (Korn and Faber, 1996).
507
ACKNOWLEDGMENTS
We are grateful to Dr. Faber (Allegheny University,
Philadelphia, PA) for useful comments throughout this
work, and to N. Ankri and J. Asmanis for computer and
secretarial help. This work was sponsored by INSERM
fellowship to K.H.
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