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J Neurophysiol 91: 2353–2365, 2004.
First published December 24, 2003; 10.1152/jn.01115.2003.
Electrophysiological and Morphological Characterization of Identified Motor
Neurons in the Drosophila Third Instar Larva Central Nervous System
James C. Choi, Demian Park, and Leslie C. Griffith
Department of Biology and Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02454-9110
Submitted 18 November 2003; accepted in final form 18 December 2003
Choi, James C., Demian Park, and Leslie C. Griffith. Electrophysiological and morphological characterization of identified motor neurons in the Drosophila third instar larva central nervous system. J
Neurophysiol 91: 2353–2365, 2004. First published December 24,
2003; 10.1152/jn.01115.2003. We have used dye fills and electrophysiological recordings to identify and characterize a cluster of
motor neurons in the third instar larval ventral ganglion. This cluster
of neurons is similar in position to the well-studied embryonic RP
neurons. Dye fills of larval dorsomedial neurons demonstrate that
individual neurons within the cluster can be reproducibly identified by
observing their muscle targets and bouton morphology. The terminal
targets of these five neurons are body wall muscles 6/7, 1, 14, and 30
and the intersegmental nerve (ISN) terminal muscles (1, 2, 3, 4, 9, 10,
19, 20). All cells except the ISN neuron, which has a type Is ending,
display type Ib boutons. Two of these neurons appear to be identical
to the embryonic RP3 and aCC cells, which define the most proximal
and distal innervations within a hemisegment. The targets of the other
neurons in the larval dorsomedial cluster do not correspond to embryonic targets of the neurons in the RP cluster, suggesting rewiring
of this circuit during early larval stages. Electrophysiological studies
of the five neurons in current clamp revealed that type Is neurons have
a longer delay in the appearance of the first spike compared with type
Ib neurons. Genetic, biophysical, and pharmacological studies in
current and voltage clamp show this delay is controlled by the kinetics
and voltage sensitivity of inactivation of a current whose properties
suggest that it may be the Shal IA current. The combination of genetic
identification and whole cell recording allows us to directly explore
the cellular substrates of neural and locomotor behavior in an intact
system.
Locomotion is a complex behavior that requires coordinated
nervous system output to multiple muscles. This coordinated
output has been shown to be the result of central pattern
generator circuits in both vertebrate and invertebrate systems
(Marder and Calabrese 1996). The output of most central
pattern generators drives motor neuron firing and can be
shaped by sensory inputs. Changes in locomotion usually involve alterations of synaptic strengths and/or the intrinsic properties of the constituent neurons. In both vertebrate spinal cord
(Kiehn 1991), where the motor neurons are not part of the
central pattern generator, and in lobster stomatogastric ganglion (Marder 1998), where they are, modulation of the properties of motor neurons is also important for adjusting output of
the central pattern generator.
In Drosophila third instar larvae, the function of central
pattern generators has been studied only by recording muscle
output (Cattaert and Birman 2001; Gorczyca et al. 1991);
circuit components have not yet been identified. The output
synapse, the third instar larval neuromuscular junction (NMJ)
has been studied intensely and is arguably the premier system
for using genetics and electrophysiology to understand synaptic development, transmission and plasticity. In the traditional
NMJ preparation, the synaptic response is measured by impaling the muscle or making a focal patch over a bouton. Typically, recordings are done with the CNS cut off so that the
contribution of the presynaptic cell is limited to that of the
distal axon and the terminal.
For understanding coordinated nervous system function, the
NMJ preparation is not ideal. In the animal, the activation of
synapses usually occurs as a consequence of an action potential
generated by integration of synaptic inputs in the somatodendritic compartment. The firing properties of the neuron, e.g.,
spike frequency, amplitude, and duration, are determined by
the balance of inward and outward currents in the cell. In
principle, this allows individual cells to develop a unique
response to inputs. Until recently (Rohrbough and Broadie
2002), third instar Drosophila motor neurons have been characterized primarily by their terminal morphology, position, and
neurotransmitter content (Anderson et al. 1988; Hoang and
Chiba 2001; Jia et al. 1993; Johansen et al. 1989; Kurdyak et
al. 1994; Monastirioti et al. 1995). Differences in the release
properties and plasticity of Ib and Is terminals have been
documented indirectly with focal patch recordings over the two
bouton types or low-level stimulation to distinguish terminal
thresholds (Kurdyak et al. 1994; Lnenicka and Keshishian
2000), suggesting that these two neurons types are functionally
different. Direct electrophysiological experiments in neurons
of the larval stage were limited to cultured, dissociated cells
(Solc and Aldrich 1988; Wu et al. 1983b) and boutons (Martinez-Padron and Ferrus 1997). In contrast, embryonic motor
neurons have been studied electrophysiologically in vivo
(Baines and Bate 1998; Baines et al. 1999, 2001) as have adult
thoracic ganglion motor neurons (Trimarchi and Murphey
1997), suggesting that access to ventral ganglion motor neurons was technically feasible. Recordings from unidentified
ventral ganglion neurons confirmed this (Rohrbough and
Broadie 2002).
In addition to answering functional questions, direct access
to intact identified motor neurons in the third instar would also
facilitate investigation of development of central circuits. Neurons of the ventral ganglion are arrayed in bilaterally symmetrical repeats that correlate with the segmental organization of
Address for reprint requests and other correspondence: L. C. Griffith, Dept.
of Biology, MS008, Brandeis University, 415 South St., Waltham, MA 024549110 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked ‘‘advertisement’’
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
INTRODUCTION
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0022-3077/04 $5.00 Copyright © 2004 The American Physiological Society
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J. C. CHOI, D. PARK, AND L. C. GRIFFITH
the body wall, and the relative orientations of neuronal somata
are stereotypic throughout segments A2–A7. During embryonic and larval development, the growth cone and varicosities
of motor neurons are in a state of constant flux (Yoshihara et
al. 1997). In the embryo, outgrowth, pathfinding, and target
recognition of a group of dorsomedial neurons (RP1– 4 and
aCC) has been extensively studied (Bate and Broadie 1995;
Broadie et al. 1993; Halpern et al. 1991; Sink and Whitington
1991b; Thomas et al. 1984). Filling of embryonic neurons
allowed the identification of initial targets. Backfilling of third
instar larval neurons from the terminal has suggested that the
position and/or targets of these embryonic neurons may change
during development of the larval CNS (Hoang and Chiba 2001;
Sink and Whitington 1991a). The final targets, central connections, and relative positions in the ventral ganglion of these
neurons need to be investigated by accessing the cell bodies.
To study the intact mature third instar CNS, we have morphologically and electrophysiologically characterized a subset
of the motor neurons that innervate the body wall muscles.
These neurons receive output from the central pattern generator
for locomotion, and their modulation is likely to be important
to plasticity of locomotor behavior. We find that significant
structural reorganization occurs between the late embryo and
the third instar. We also find that there is diversity in the
intrinsic properties of larval motor neurons and that differences
in potassium current regulation can control the cell-specific
properties of identified neurons.
METHODS
Preparation
All experiments were performed on wandering, primarily female,
third instar larvae. Animals were dissected and pinned in 0.2 mM
Ca2⫹ “A” solution (which contained, in mM, 128 NaCl, 2 KCl, 35
sucrose, 4 MgCl2, and 5 HEPES, pH 7.1–7.2 (original solution contains 1.8 mM Ca2⫹) (Jan and Jan 1976), with care taken to leave
segmental nerves and the CNS intact. Calcium-free “A” solution was
used as the external solution for all experiments with the exception of
the confocal dye-fill experiments (see details in the following text).
The preparation was visualized using an upright Olympus microscope
equipped with differential interference contrast and epifluorescence.
Under constant perfusion with laminar flow traversing, the larva
toward the anterior direction, collagenase type I or protease XIV (1
and 2 mg/ml, respectively, Sigma-Aldrich, St. Louis, MO) was focally
administered to the ventral ganglion using a glass pipette pulled to
⬃10 ␮m tip diameter. Debris was constantly cleared using the pipette
with positive and negative pressure. With adequate exposure, the
outer layer of the ganglion is disrupted, revealing neural tissue.
Prominent somata with diameters of 10 –15 ␮m could be seen at this
point. The protease treatment was continued until an inner membrane
layer enclosing the group of neurons was disrupted. A successful
procedure rendered the soma clean, smooth and loose, but intact
within its cluster. Recordings were performed mainly in segment A3.
Genetics, cell visualization, and identification
Motor neurons were visualized using GFP fluorescence. GFP expression was driven by C164-GAL4 (a generous gift of Vivian Budnik, University of Massachusetts, Amherst, MA), an enhancer trap
line that expresses in all motor neurons (Packard et al. 2002). Crossed
with UAS-mCD8-GFP, C164-GAL4 allows sharp, distinct visualization of cells, as mCD8 targets the GFP to the membrane (Lee and Luo
1999). After dissection and protease digestion, the targeted soma was
J Neurophysiol • VOL
approached with constant positive pressure until the pipette tip was
apposed to the surface. After establishing a tight seal, the membrane
was broken with instantaneous suction and/or current injection. The
recording intracellular solution usually contained tetramethyl rhodamine dextran 3000 (10 mg/ml, Molecular Probes, Eugene, OR), either
backfilled into the pipette or as part of the solution, which gave a
slightly lower patch success rate but did not alter neuronal responses
(data not shown). After ⬃15 min of filling, during which physiological recordings were obtained, the processes and terminal morphology
of the targeted neurons were visualized on the same microscope.
Images were taken with a Photometrics PXL liquid-cooled CCD
camera and IPLab software package on a Macintosh computer. Combinations of brightfield, GFP, and dye images were rendered using
NIH Image and Adobe Photoshop.
For every experiment, a specific soma within a stereotypical cluster
in the dorsomedial surface was targeted, relying on its orientation
relative to the midline and the other somata. ShKS133, a point mutation
in the Sh gene (Lichtinghagen et al. 1990) that eliminates IA in muscle
(Solc et al. 1987; Wu et al. 1983a) was used to dissect the IA
potassium current. In this case, DIC and not GFP was used to target
neurons for recording, and the identity of the neuron was confirmed
with dye fill.
For detailed confocal visualization of axon and dendrites of individual motor neurons using confocal microscopy, larvae were dissected in HL3 (0 mM Ca2⫹), and the recording intracellular solution
contained a 10:2 mixture of tetramethyl rhodamine dextran 3000 (10
mg/ml) and AlexaFluor 568 hydrazide sodium salt (10 mg/ml, Molecular Probes). After 30 – 60 min of filling, larvae were fixed in
phosphate-buffered 4% paraformaldehyde for 30 min at room temperature. The larval tissues were then washed in HL3, followed by 30,
60, and 100% glycerol incubations for 10 min prior to mounting in
Vectashield (Vector Laboratories, Burlingame, CA). Confocal images
were acquired using a Leica confocal system. Images were then
further processed in Adobe Photoshop.
Electrophysiology
Filamented thin- and thick-walled capillary pipettes (WPI, Sarasota, FL) were pulled and fire polished to a resistance of 5–10 M⍀.
Intracellular recording solution (adapted from O’Dowd 1995) contained (in mM) 120 potassium gluconate, 20 KCl, 10 HEPES, 1.1
EGTA, 2 MgCl2, and 0.1 CaCl2. The pH was adjusted to 7.2 and the
osmolarity to 280 mmol/kg. Recordings were performed with an
Axopatch 200B amplifier (Axon Instruments, Foster City, CA) and
ITC-16 data acquisition board (National Instruments, Austin, TX)
with Igor software (Wavemetrics, Lake Oswego, OR). Cell membrane
potentials were recorded in whole cell current-clamp mode. Spikes
were evoked by a series of current injections delivered via the recording pipette. The duration of current steps was 500 or 1,000 ms, and the
amplitude ranged from –20 to 140 pA in 20-pA increments. In
prepulse experiments under current clamp, the external solution contained 0 mM Ca2⫹. The duration of the prepulse was 1 s, and the
amplitude was ⱖ20 pA below spike threshold, usually 20 pA. The
average prepulse depolarization was to – 43 ⫾ 1 mV (n ⫽ 8 cells). The
prepulse was immediately followed by test current steps of 1-s duration. In experiments to pharmacologically eliminate transient potassium currents, 4-aminopyridine (4-AP; 1.5 mM, Calbiochem, San
Diego, CA) was bath-applied. Recordings were obtained before and
after application, and also 2 min after several washes of external
solution. For recordings of spontaneous activity, 2.4 mM Ca2⫹ was
added to the external solution, and passive recordings were done in
whole cell configuration.
In neuron-muscle dual recordings, EJPs were recorded using sharp
electrodes (⬃30 M⍀) filled with 3 M KCl for muscle. Having
achieved whole cell access to the neuron, the preparation was visualized under lower magnification (⫻10), and the corresponding muscle was impaled with a recording electrode. The external Ca2⫹ con-
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VENTRAL GANGLION MOTOR NEURONS
centration was increased to 0.4 mM. Under current clamp, a stimulus
protocol was performed on the neuron and recordings made simultaneously from neuron and muscle. EJPs were amplified with Axoclamp
2B (Axon Instruments), which was connected to the same acquisition
board.
For voltage-clamp recordings, neurons were switched to voltageclamp mode after initial current-clamp recordings. Sodium channels
were blocked with tetrodotoxin (Sigma, 100 nM). Cells were held at
either – 80 or –100 mV. For trace subtractions, voltage was raised to
⫹40 mV for 1 s, and the difference between the waveform obtained
and one recorded from a holding potential of – 40 mV was calculated.
For current-voltage data, test pulses ranging from –120 to ⫹40 mV (in
20-mV increments) were given in 15-s intervals. Waveforms obtained
with the –20 mV pulse was used to subtract leakage currents. To
obtain voltage-dependent inactivation plots, a 1-s prepulse of variable
amplitude (⫺100 to ⫹40 mV in 10-mV increments) was given, and a
test pulse of ⫹40 mV amplitude was given after a 10-ms interpulse
interval. Perfusion was usually stopped for voltage-clamp recordings.
Identity of neurons was subsequently verified by visualizing the dye
fill. Capacitance normalization was done with values obtained directly
from the parameter settings on the amplifier.
Series resistances of cells ranged from 20 to ⬎100 M⍀, most likely
due to clogging of the pipette tip by cellular constituents. As an
acceptance criterion, only experiments where Rin ⬎1 G⍀ and calculated maximum voltage error ⬍25 mV were analyzed.
Data analysis
For injection of different levels of current, time to peak was
measured as the time between the beginning of current injection to the
peak of the first spike observed in the voltage trace. Peak amplitude
was derived by averaging the amplitude of all the spikes in a given
voltage trace. Spike frequency was calculated by averaging the instantaneous frequencies derived by measuring the time interval between adjacent spikes. Only the first 500 ms of each trace was
analyzed. The inactivation time constant (␶) was obtained by using a
single exponential fit from peak to steady state for each waveform. For
the inactivation plot, the peak was normalized with the maximum and
minimum values, and plotted with respected to prepulse amplitude. A
regular Boltzmann equation was used to fit the plot, and the halfmaximal inactivation voltage was obtained.
Statistical analyses were done in Statview 4.5 (Abacus Concepts/
SAS, Carry, NC) or the analysis tools package in Excel (Microsoft,
Redmond, WA). P values reported for multiple comparisons were ascertained using ANOVA with Scheffe’s post hoc test for significance.
RESULTS
Identification of dorsomedial motor neuron projections and
targets in the third instar larva
Using the standard NMJ dissection, neurons in the ventral
ganglion can be readily visualized and particular groups of
neurons identified using cell-specific GAL4 drivers. We have
used C164-GAL4 (Packard et al. 2002) to drive expression of
GFP in motor neurons to target our studies to this population.
The motor neurons in the ventral ganglion are organized stereotypically (Sink and Whitington 1991a) with segmental arrays of somata repeating along the anterior-posterior axis.
Axons project to the body-wall muscle through nerves that
emanate from the ganglion in each hemisegment. We focused
on the bilaterally symmetric dorsomedial clusters that are
aligned parallel to the midline. Figure 1A shows GFP expression in the posterior end of a preparation after dissection. A set
of five neurons that are GFP positive repeats in segments
A2–A7 with all the neurons lying in the same plane of view.
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Figure 1, B and C, shows brightfield and GFP visualizations of
the dorsomedial cluster at higher magnification after enzymatic
digestion. The somata are typically in a cross formation, although shifts in relative orientation can cause some deviation
from this pattern.
The identities of the neurons in the dorsomedial cluster were
determined by filling GFP-positive cell bodies from the recording patch pipette, which contained rhodamine and/or Alexa
568. After recording, the body-wall muscles were imaged for
GFP and dye. Each fill resulted in dye signal in a single axon
and bouton arbor that could be visualized in the live preparation. Correlation of soma position, muscle target, and bouton
morphology suggests that innervation type and target are invariant for each cell within the cluster. Each neuron has an
elaborate projection pattern within the ventral ganglion that is
always in the same relative position. Example fills of each of
the five neurons are shown in Fig. 1, D–H, and a schematic of
the arborization patterns is shown in I.
To name the neurons, we have adopted the nomenclature
devised by Hoang and Chiba (2001), naming each neuron
according to its target muscle and the morphology of its terminal boutons. The five neurons of the dorsomedial cluster
were identified as MN1-Ib, MN6/7-Ib, MN14-Ib, MN30-Ib,
and MNISN-Is. Table 1 provides a summary of the cell’s
morphological features. Bouton types were categorized primarily by relative size compared with the other boutons in the field
of view, and their consistency with previously published accounts of muscle innervations (Hoang and Chiba 2001). Figure
2 shows the muscle termini of each of the five neurons. A
schematic of the organization of the muscles and nerves in one
hemisegment is shown in Fig. 2A.
Four of the five neurons in the dorsomedial cluster terminate
with type Ib boutons. MN14-Ib and MN30-Ib are unipolar and
project contralaterally through the segmental nerve (Fig. 1, E
and G). Axons from these two neurons travel together until the
terminal arborization point in SNb (segmental nerve branch b)
where they diverge to innervate their respective neighboring
muscle targets, 14 (Fig. 2D) and 30 (Fig. 2E). MN6/7-Ib
projects bilaterally in the ventral ganglion, but a single process
extends contralaterally past the midline and out through the
segmental nerve, terminating exclusively with type Ib boutons
on muscles 6 and 7 (Figs. 1F and 2C). MN1-Ib has prominent
bilateral projections with two dendritic arborizations within the
ventral ganglion (Fig. 1D); the motor axon projects ipsilaterally through the ISN (intersegmental nerve) to innervate the
distal muscle 1 (Fig. 2B).
The anterior-most neuron in the cluster is MNISN-Is, which
projects ipsilaterally through the ISN. The axonal process
climbs up and exits the nerve of the anterior segment, so that
its muscle innervation is actually one segment anterior to those
of other neurons within the cluster (Fig. 1H). This neuron targets
multiple muscles on the distal (dorsal) body wall (Fig. 2F). The
identification of this neuron as a Is neuron was supported by the
fact that in muscle 4, in view of the slightly bigger boutons of
Ib, the dye-filled innervating bouton type was clearly type Is
and not type II (Fig. 2F). We unambiguously observed innervation of muscles 1, 2, 3, 4, 9, 10, 19, and 20, and muscle 18
may also have innervation that was less clear and is not seen in
Fig. 2. Hoang and Chiba describe a neuron (MNISN-Is) that
innervates muscles 1, 2, 3, 4, 9, 10, 18, 19, and 20 (Hoang and
Chiba 2001). This is almost the exact target pattern of our
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J. C. CHOI, D. PARK, AND L. C. GRIFFITH
FIG. 1. Ventral ganglion projections of dorsomedial motor neurons. A: a low-power (⫻10) view of the abdominal segments of
a 3rd instar ventral ganglion expressing mCD8-GFP under control of C164-GAL4. Motor neuron clusters are seen in each
hemisegment. B and C: a close-up of 2 segmental clusters using DIC (B) and fluorescence (C). Scale bar ⫽ 25 ␮m for B–F; 6 ␮m
for A; a, anterior and p, posterior. The midline is denoted by a dotted line. D–H: dye fills of each neuron. Insets: a lower
magnification of the same image with the GFP signal overlaid and the midline noted by a dotted line. D: MN1-Ib; E: MN14-Ib;
F: MN6/7-Ib; G: MN30-Ib; H: MNISN-Is. I: a schematic of the cell body location and projections of the dorsomedial motor
neurons.
observation of MNISN-Is. Hoang and Chiba (2001) note that
all muscles that are innervated by the ISN most likely receive
Is innervation from a single neuron. This is quite different from
type Ib innervations which usually arise from neurons dedicated to a single muscle or pair of muscles.
On three occasions, in abdominal segment 2, we observed a
type Is innervation of muscles 6/7. This neuron also innervated
additional muscles with a type Is bouton (Table 1). The muscle
pattern was consistent with Hoang and Chiba’s MNSNb/d-Is.
The cell body of this neuron was located beneath the cluster of
five dorsomedial neurons that we have more fully characterized
but could not be consistently targeted as the cell body is not
predictably in the superficial cluster.
TABLE
Dye fills established anatomical connections between dorsomedial neurons and specific muscle targets. To demonstrate
that these neuromuscular connections were functional and
could be used to address questions of individual neuron function, neuron-muscle dual recordings were done. Figure 3Gi
shows paired recordings from MN6/7-Ib and muscle 6. Each
neuronal action potential reliably evoked an EJP in the target
muscle. Spontaneous patterned activity within the ventral ganglion can also be observed when the extracellular solution has
a high concentration of calcium. Figure 3Gii shows a currentclamp trace of MNISN-Is in 2.4 mM calcium. The frequency of
bursting is similar to what has been previously identified for
the locomotor pattern generator (Cattaert and Birman 2001).
1. Morphology of third instar dorsomedial motor neurons
Third Instar
Motor Neuron
Embryonic
Identity
Nerve
Target Muscles
Projections
n
MN1-Ib
MNISN-Is
MN14-Ib
MN6/7-Ib
MN30-Ib
MNSNb/d-Is
aCC
RP2
RP1 or RP4?
RP3
RP1 or RP4?
RP5?
ISN
ISN
SN
SN
SN
SN
1
1, 2, 3, 4, 9, 10, (18), 19, 20
14
6/7
30
6, 7, 13, 14, 30, (15, 16)
Ipsilateral axon, major contralateral process
Ipsilateral axon
Contralateral axon
Contralateral axon, major ipsilateral process
Contralateral axon
Contralateral axon
6
17
20
12
14
3
The structure and targets of dorsomedial neurons were investigated in C164-GAL4; UAS-mCD8GFP animals by filling from the cell body with rhodamine.
Nomenclature is adopted from Hoang and Chiba (2001) and indicates the muscle(s) innervated and the bouton type. Target muscles in parentheses were not
examined but were seen by Hoang and Chiba (2001). For MNISN-Is, the target muscles listed are cumulative observations; not all muscles were observed in
every preparation. Projections refers to the processes seen within the ventral ganglion. The number of preparations examined is indicated by n. ISN,
intersegmental nerve; SN, segmental nerve.
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VENTRAL GANGLION MOTOR NEURONS
Evoked spiking activity in dorsomedial larval neurons
The dorsomedial neurons show consistent position, target,
and morphology from animal to animal. To determine if firing
properties were also determined by cell identity, we recorded
evoked action potentials from the somata of these neurons.
Current injection readily caused repetitive spikes at frequencies
that were dependent on stimulus intensity. Hyperpolarizing
prepulses did not evoke significant activity (data not shown).
Figure 3, A-F, shows representative traces from current-clamp
recordings of the five neurons within the cluster and a recording from a putative MNSNb/d-Is cell. Data acquisition was
typically done as the cells were being filled with dye; this
allowed unequivocal identification of the neurons and correlation of morphology with physiology. Resting membrane potential, resting input resistance and current required for spiking
are shown in Table 2. The amount of current required (in
20-pA increments) to initiate spiking was not different comparing the Ib neurons to each other, but MNISN-Is required
significantly more current than any of the Ib cells (P ⬍ 0.001).
Data for time to first spike for each of the five cells are
shown in Fig. 3H. The MNISN-Is neuron had a significant
delay to spiking compared with the Ib neurons (Fig. 3H, P ⬍
0.0001 for all current injections). Time to first spike was not
different between Ib neurons at any current injection level (Fig.
3H, P ⬎ 0.35).
Mechanism of the MNISN-Is delay
Because the identity and properties of the larval motor
neurons are stereotyped, the molecular basis of the behavior of
each neuron can be addressed using pharmacological, biophysical, and genetic tools. A distinct structural feature of
MNISN-Is and MNSNb/d-Is is that, unlike the type Ib neurons,
they innervate multiple muscles. The striking delay in the time
to first spike is likely to have effects on patterned muscle
activity. In other neurons where such delays have been observed, they have often been due to the presence of an inactivating potassium current (Turrigiano et al. 1996; Zhao and Wu
1997). To determine if this was the mechanism underlying the
MNISN-Is firing kinetics, we examined the effects of depolarizing prepulses and 4-AP on the delay. Both of these manipulations are known to decrease IA-type currents. Figure 4A
shows traces from an MNISN-Is cell before, during and after
washout of 1.5 mM 4-AP. During exposure to the drug, there
is a decrease in the time to first spike that is reversed on
washout. 4-AP also affects spiking behavior; stereotyped, regular single spike activity is altered to give doublet spikes. In the
example shown in Fig. 4A, the doublet spiking is also seen in
the wash, but in other cells this effect of 4-AP did wash out
(data not shown). Depolarizing prepulses delivered prior to
suprathreshold test amplitudes had an effect similar to 4-AP
(Fig. 4C), decreasing the time to spike onset. The mean level of
depolarization achieved with prepulse was – 43 ⫾ 1 mV (n ⫽
8). Prepulse also caused doublets in the spike train, but the
effect was less consistent, with multiple spike activity appearing sporadically during test pulse (data not shown). Subsequent
experiments with longer test pulse protocols in control cells
revealed that with long current injections (⬎2 s), even control
cells could show doublet spiking.
2357
The changes in delay with 4-AP and prepulse are quantified
in Fig. 4, B and D, for different levels of current injection. The
decrease in time to first spike was significant for comparison of
control and washout with 4-AP for all current injections (P ⬍
0.05). Control and washout were not significantly different,
except at the lowest stimulus amplitude (P ⬎ 0.02). For prepulse experiments, control and prepulse delays were significantly different at all current spike-evoking injection levels
(P ⬍ 0.01).
To determine the identity of the 4-AP- and prepulse-sensitive potassium current(s) responsible for the delay in onset of
spiking and doublet spiking, we examined the firing behavior
of MNISN-Is in ShKS133 mutants. The Sh gene encodes one of
the channel proteins known to produce IA. In Sh mutants, the
fast inactivating current of third instar larval muscles is selectively eliminated (Wu and Haugland 1985; Wu et al. 1983a).
The other gene known to encode an IA channel is shal. The
Shal protein is thought to be responsible for the majority of IA
in embryonic neurons (Scholz et al. 1988; Tsunoda and Salkoff
1995). A comparison of the delay in MNISN-Is recordings
from ShKS133 (Fig. 4E) and control (Fig. 4A) neurons shows
that the magnitude of delay is not significantly different for any
spike-inducing current injection level (P ⬎ 0.1). In all of the
recordings from Sh mutant neurons, doublet spikes were seen
in the spike train (Fig. 4E). These doublets were qualitatively
similar to those seen with 4-AP treatment and prepulse. Prepulse of MNISN-Is in Sh mutants is still able to decrease the
time to first spike, suggesting that there is a non-Shaker prepulse-sensitive current that causes the delay. A comparison of
the delay before and after prepulse for different current levels
is shown in Fig. 4F. For all levels of current injection, the
decrease in the delay ranged from 80 to 95%, and Sh was
significantly different from Sh with prepulse (P ⬍ 0.05 for all
current injection levels). These results suggest that the role of
Shaker in the MNISN-Is neuron does not involve setting the
spike delay. Spike delay is mediated by another prepulsesensitive current, likely carried by Shal. Unfortunately, beTABLE
2. Properties of third instar dorsomedial motor neurons
Motor Neuron
Resting Input
Resistance,
M⍀
Resting
Membrane
Potential, mV
Average Current
Injection to
Evoke Spikes,
pA
n
MN1-Ib
MN6/7-Ib
MN14-Ib
MN30-Ib
MNISN-Is
977 ⫾ 89
1204 ⫾ 79
1322 ⫾ 127
1435 ⫾ 70
1653 ⫾ 172*
⫺56 ⫾ 1
⫺58 ⫾ 2
⫺54 ⫾ 1
⫺58 ⫾ 2
⫺61 ⫾ 2
27 ⫾ 5
32 ⫾ 4
26 ⫾ 4
29 ⫾ 4
74 ⫾ 7**
6
12
10
7
10
Electrophysiological properties of dorsomedial neurons were measured in
whole cell current-clamp mode. Resting input resistance was measured by
potential difference with a ⫺20-pA current pulse. MNISN-Is was had a
significantly higher resting input resistance compared to MN1-Ib (*, P ⬍ 0.05).
Spikes were induced by current injections from 20 to 140 pA in 20-pA
increments. The level of current injection that 1st induced spiking was noted
for each cell. There was no significant difference in the average current
required to induce spiking in Ib neurons (P ⬎ 0.05). MNISN-Is required
significantly more current than any of the Ib neurons (**, P ⬍ 0.001). There
was no difference in resting membrane potential among all the neurons
(P ⬎ 0.05). The number of cells of each type characterized is indicated by n.
Values are means ⫾ SE.
2358
J. C. CHOI, D. PARK, AND L. C. GRIFFITH
VENTRAL GANGLION MOTOR NEURONS
2359
FIG. 3. Firing properties of dorsomedial neurons are stereotyped. Whole cell current-clamp recordings were made from
identified motor neurons. Spiking was evoked with injection of current from –20 to 140 pA in 20-pA increments for 500 ms except
for F where the duration was 1 s. For clarity, only traces from the –20-, 0-, 20-, 60-, and 100-pA injections are shown. A–F:
representative examples of each motor neuron type. Resting membrane potential for the example cells was: MN1-Ib, ⫺54;
MN6/7-Ib, ⫺59; MN14-Ib, ⫺58; MN30-Ib, ⫺55; MNISN-Is, ⫺58; and MNSNb/d-Is, ⫺55. Gi: that the connection between
MN6/7-Ib and its target muscle 6 is functional. Simultaneous neuron and muscle recordings show time locked neuronal spikes and
muscle EJPs. Gii: patterned synaptic activity onto MNISN-Is in 2.4 mM external calcium. H: graph of time to 1st spike for each
neuron. MNISN-Is is significantly different from the rest of the neurons (P ⬍ 0.0001 for all current injections). The scale bars for
A–E are 20 mV and 200 ms; for F and Gi, they are 400 ms and 20 mV for neuronal trace and 0.2 mV for the muscle EJPs; for
Gii, they are 5 s and 10 mV.
cause there are no shal mutants, the role of Shal cannot be
directly approached genetically.
Mechanism of delay in Ib neurons
The Ib motor neurons have a very short delay to first spike.
To determine if this delay was due to a Sh encoded IA, we
measured the delay in wild-type and ShKS133 neurons. Figure
4G shows the delay to first spike measured after a 60-pA
current injection. None of the Ib neurons have a statistically
significant decrease in delay when Sh is mutated (P ⬎ 0.05).
We also examined the effect of Sh mutation on spike rate and
saw no significant differences in any of the neurons for current
injections of 60 –140 pA (P ⬎ 0.3). These data suggest that in
Ib neurons, as in MNISN-Is, the Shaker channel does not play a
significant role in determination of spike rate or delay to first spike.
Isolation of potassium currents in larval motor neurons
To characterize the potassium currents present in larval
motor neurons, we recorded outward currents from MNISN-Is
and MN6/7-Ib in voltage clamp. Figure 5 shows example
whole cell recordings and peak (solid line) and sustained
(broken line) current I-V curves. Current densities were similar,
as were current voltage relationships, indicating that the activation properties of the outward currents in Ib and Is neurons
are similar.
The fast inactivating component of the whole cell outward
current was isolated by subtracting currents obtained with a
pulse to ⫹40 mV after holding at – 40 mV from total currents
obtained after holding at –100 mV. Figure 6, A and B, shows
example subtracted currents from MNISN-Is and MN6/7-Ib
neurons. Figure 6C shows averaged subtracted currents for
FIG. 2. Identification of dorsomedial motor neuron targets. The targets of the dorsomedial neurons were identified by patching
onto and filling GFP-positive neurons with rhodamine/Alexa 568 and following the axons onto the segmental muscles. A: a
schematic of the relevant body-wall muscles of a single abdominal hemisegment. Muscles targeted by segmental nerve b/d (SNb/d)
are shown in red, and those targeted by the intersegmental nerve (ISN) in blue. Muscles innervated by the dorsomedial motor
neurons are shown in lighter tone. B–F: overlayed GFP and dye-fill images. B: MN1-Ib; C: MN6/7-Ib; D: MN14-Ib; E: MN30-Ib;
F: MNISN-Is. Single arrow, bouton type Ib; double arrows, bouton type Is; and triple arrows, bouton type II. Scale bar ⫽ 25 ␮m
for B–E, 10 ␮m for F.
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J. C. CHOI, D. PARK, AND L. C. GRIFFITH
FIG. 4. Delay to spike in MNISN-Is is due to a
prepulse- and 4-aminopyridine (4-AP)-sensitive nonShaker current. A: traces from MNISN-Is before 4-AP
application (top), during application of 1.5 mM 4-AP
(middle), and after washout (bottom). B: summarization of the time to st spike for each condition for
current injections of 60 –140 pA. Control and washout
delays were significantly different from 4-AP (P ⬍
0.05). For 4-AP, n ⫽ 4. C: traces from MNISN-Is
without (top) and with (bottom) a 1-s depolarizing
prepulse. D: the time to 1st spike for each condition
for current injections of 60 –140 pA. Control and
prepulse were significantly different (P ⬍ 0.01). For
prepulse, n ⫽ 4. D: traces recorded from MNISN-Is in
a ShKS133 mutant larva. Bottom: the effects of a 1-s
depolarizing prepulse. F: summarization of the time to
1st spike for each condition for current injections of
60 –140 pA. Control and prepulse for Sh were significantly different (P ⬍ 0.01). For Sh neurons, n ⫽ 4. G:
summarization of the time to 1st spike data comparing
wild-type and Sh neurons for all neuron types with a
60-pA current injection. None of the differences are
significant (P ⬎ 0.05).
multiple neurons (n ⫽ 6 for MNISN-Is and n ⫽ 4 for MN6/
7-Ib). Although the amplitude of the prepulse-sensitive current
is similar, the rate of decay was significantly different for the
two neuron types, with a ␶ of 122 ⫾ 22 ms for MNISN-Is and
54 ⫾ 12 ms for MN6/7-Ib (P ⬍ 0.05).
Inactivation rate may contribute to the cell-specific difference in spike delay, but it is not likely to completely account
for the magnitude. Using a range of prepulse amplitudes, we
investigated the voltage dependence of prepulse inactivation in
MN6/7-Ib and MNISN-Is. The normalized peak amplitudes of
the resultant currents were plotted with respect to prepulse voltage
amplitude, and a Boltzmann fit was applied. Figure 7 shows that
J Neurophysiol • VOL
there is a clear difference in the voltage dependence of inactivation. At a membrane potential of – 60 mV, the amplitude of IA
in MNISN-Is is at ⬃90%, while for the MN6/7-Ib it is ⬍50%. At
normal resting membrane potential, the activity of the transient
current will therefore have a greater role in the MNISN-Is.
DISCUSSION
Relationship of the larval dorsomedial cluster to the
embryonic RP cluster
While much is known about the proliferation and organization of specific neurons in the embryonic stage, only a few
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VENTRAL GANGLION MOTOR NEURONS
FIG. 5. Total outward currents in MNISN-Is and MN6/7-Ib are equivalent.
Outward currents were recorded in voltage clamp in the presence of TTX and
0 mM calcium. Cells were held at Vm ⫽ – 80 mV and stepped to different
potentials in 20-mV increments. A and B: sample traces of whole cell currents
obtained from MN6/7-Ib and MNISN-Ib, respectively. Vertical scale bar is 50
pA; horizontal bar is 200 ms. C: I-V curves of average peak (solid lines) and
steady-state (dashed lines) amplitudes of the outward currents in the MNISN-Is
and MN6/7-Ib neurons. Error bar indicates SE. n ⫽ 5 for MN6/7-Ib and 6 for
MNISN-Is.
studies have detailed the subsequent developmental transformations that occur between the late embryonic stage and the
third instar larval stage (Hoang and Chiba 2001; Landgraf et al.
2003; Schmid et al. 1999; Sink and Whitington 1991b). It is
2361
apparent that the organism undergoes significant morphological adjustments as the ventral ganglion retracts toward the
anterior, and the larva grows exponentially in size and complexity. It is likely that considerable modification of the neural
circuitry is also taking place. The dorsomedial cluster of neurons in the third instar ventral ganglion that we have observed
bears a striking resemblance to the cluster of embryonic neurons containing aCC and RP1– 4 with respect to spatial orientation. Like the larval cluster, the aCC and RP neurons are the
most dorsal of the ganglion’s neurons, and the relative configurations of the somata and axonal processes seem to be generally consistent. Two of the embryonic neurons, aCC and
RP3, appear to be wholly conserved in terms of projection and
connectivity, corresponding to larval MN1-Ib and MN6/7-Ib,
respectively. The identity of MN6/7-Ib as RP3 agrees with the
findings of Hoang and Chiba (2001), who backfilled cell bodies
from individual muscle terminals. In their study, there were no
landmarks within the ventral ganglion to orient the cells with
respect to each other, but the medial location and contralateral
projection of MN 6/7-Ib, as well as its parallel dendritic arborizations, is well documented. The aCC neuron appears to be
maintained as MN1-Ib, which uses an ipsilateral projection to
connect to muscle 1. Both aCC and MN1-Ib have a major
contralateral projection that terminates within the ventral ganglion as well as dendritic arborizations concentrated around the
ipsilateral axonal tracts (Landgraf et al. 1997; Schmid et al.
1999). Single nerve backfills of embryonic and larval preparations showed that an embryonic neuron identified as aCC has
a conserved larval counterpart that is directly lateral to the
posterior of a group of cells resembling the RP cluster.
MN14-Ib, MN30-Ib, and MNISN-Is have muscle targets that
do not match those of the embryonic RP neurons. RP1 and RP4
project contralaterally to innervate the proximal edge of muscle
13 in the embryo. MN14-Ib and MN30-Ib also have contralateral projections, but they go to different targets. If they are the
FIG. 6. Isolated IA potassium currents in MNISN-Is and
MN6/7-Ib. A and B: sample traces of subtracted currents obtained in voltage clamp. A test pulse to ⫹40 mV was given with
the neuron held at – 40 and –100 mV. Currents from the former
were subtracted from the latter to isolate prepulse-sensitive
currents. Vertical scale bar is 2 pA/pF; horizontal bar is 200 ms.
C: averaged and normalized traces of the subtracted waveforms. n ⫽ 4 for MN6/7-Ib; n ⫽ 6 for MNISN-Is. t1/2 of
inactivation is 27 ms for MN6/7-Ib and 71 ms for MNISN-Is.
J Neurophysiol • VOL
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J. C. CHOI, D. PARK, AND L. C. GRIFFITH
FIG. 7. Voltage dependence of IA inactivation. Vm of
MN6/7-Ib (n ⫽ 3) and MNISN-Is (n ⫽ 3) were stepped
to a 1-s prepulse of –100 to 0 mV amplitude before
given a 1-s test pulse at ⫹40 mV. Normalized peak
amplitude of transient outward current was plotted with
respect to prepulse voltage amplitude. The Boltzmann fit
of the averaged values is shown. V1/2 for inactivation
was – 61 mV for MN6/7-Ib and – 48 mV for MNISN-Is.
third instar incarnations of RP1 and RP4, it would imply that
there has been retargeting between first and third instar. This
possibility is supported by the fact that the arborization points
for muscles 14 and 30 are in closest proximity to that of muscle
13; retraction of filopodia for RP1 and RP4 would place them
at the appropriate branch point location. An alternate explanation would be that the MN14-Ib and MN30-Ib are new neurons
and the cell bodies of RP1 and RP4 have shifted with respect
to the location of RP3.
The third instar MNISN-Is is likely to correspond to the
embryonic NB4 –2-derived RP2 neuron. These two neurons
both have somata that are one segment posterior to the rest of
the dorsal cluster that project through the same nerve, and also
have significant arborizations in the ipsilateral axonal fascicles.
MNISN-Is innervates the majority of dorsal muscles that are
targeted by the ISN (Table 1). In support of this identification,
Landgraf et al. (2003) show comprehensive innervation of the
dorsal muscles by RP2 in the third instar as identified by
eve-Gal4RRK expression.
The ventral location of RP5 makes it an unlikely candidate
to be any of the third instar dorsomedial cluster neurons (Sink
and Whitington 1991a). However, on three occasions, we filled
neurons with the MNSNb/d-Is innervation pattern (Table 1)
that also had physiological characteristics similar to MNISN-Is
(Fig. 3F; see following text). Although the organization of the
intraganglionic projections were not recorded, observation of
the ventral location of the soma and its medial position relative
to the five neuron cluster makes it a possible RP5 derivative.
RP5 has been observed on different occasions to innervate
muscles 12 (Landgraf et al. 1997),13, 15, 16, and 17 (Sink and
Whitington 1991a) in earlier embryonic stages and muscles 6,
7, 12, 15, and 16 in late stage 17 (Schmid et al. 1999).
Our results support findings by others (Hoang and Chiba
2001; Sink and Whitington 1991b) that location of motor
neurons and their axonal projections undergo major remodelJ Neurophysiol • VOL
ing in the interval between the late embryonic and third instar
stages. It may be significant that RP3 and aCC, the two neurons
whose morphological character seem most conserved throughout development, are among the earliest motor neurons to
differentiate, and that their targets are respectively the most
proximal and distal longitudinal muscles to the midline. These
two neurons may be conserved throughout development to be
used as templates or guideposts in the formation and maintenance of the segmental organization of the neurons in the
ventral ganglion and the axonal targeting of muscle innervation
(Lin et al. 1995).
Intrinsic properties of ventral ganglion neurons are dictated
by cell identity
The MNISN-Is and MNSNb/d-Is cells are outstanding
among the dorsomedial motor neurons in threshold and spiking
behavior, requiring more current to evoke spiking (Table 2)
and having an appreciable delay between depolarization and
the initiation of spiking (Fig. 3H). The delayed spiking phenomenon has been seen in other systems, where it has been
shown that voltage-activated transient potassium currents (IA)
can cause a delay in depolarization toward threshold (Turrigiano et al. 1996; Zhao and Wu 1997). IA is also known to be
important in the rendering of spike trains (Connor and Stevens
1971; Rogawski 1985) and has been shown to be the target of
neuromodulators that affect motor neuron function (HarrisWarrick et al. 1998). The delay in MNISN-Is is prepulse (Fig.
4C) and 4-AP (Fig. 4A) sensitive; both of these manipulations
are known to affect voltage-activated transient potassium currents IA (Solc and Aldrich 1988), suggesting a role for this
current in the behavior of the type Is neurons.
Two fast-inactivating potassium channels are known to exist
in Drosophila: Shaker and Shal. In studies of heterogeneous
populations of cultured Drosophila embryonic neurons, a small
91 • MAY 2004 •
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VENTRAL GANGLION MOTOR NEURONS
fraction were found to have both Shaker and Shal currents
(Tsunoda and Salkoff 1995). Recordings from Sh mutants
showed that the Shaker current does not have a significant role
in soma-evoked spiking behavior (initial delay or rate) in any
of the five motor neurons in this study. Thus the IA current that
controls the delay to first spike is likely to be Shal in both type
Is and Ib neurons.
How then are the behaviors of type Is and Ib neurons
differentiated? Distinct activation kinetics or amplitude differences are unlikely to underlie the cellular phenotypes. The I-V
relationships for both the fast-inactivating and sustained outward currents were not statistically different (Fig. 5C). Using a
prepulse subtraction protocol, we isolated the fast-inactivating
potassium currents in MNISN-Is and MN6/7-Ib and found
them to be similar in amplitude (Fig. 6, A and B). The major
differences between the IA currents in the two cell types was in
their inactivation. IA currents in MNISN-Is had a significantly
longer time course of inactivation. This effect alone, while
substantial, is unlikely to account for the magnitude of the
difference in time to first spike in the two neuron types. An
investigation of the voltage dependence of prepulse inactivation revealed a significant rightward shift for the MNISN-Is IA
as compared with that in MN6/7-Ib (Fig. 7). At the average
resting potential (⫺61 mV) of MNISN-Is cells, the Shal current shows essentially no inactivation. In type Ib neurons,
where resting membrane potential ranges from –58 to –54 mV,
the current is already almost completely inactivated. Together,
these differences in inactivation are likely to account for the
disparity in the time to first spike that is seen between type Ib
and Is motor neurons. There are also likely to be other currents
(sodium, calcium, chloride) that are important determinants of
the cell-specific properties of the type Ib and Is neurons, and
their roles remain to be determined.
If both type Is and Ib neurons use Shal gene products to
produce IA, the differences in inactivation are likely to involve
alternative splicing, posttranslational modification of Shal
channels or differences in auxiliary subunit expression. The
Shal/Kv4 family is highly conserved across species (Baldwin
et al. 1991; Baro et al. 1996; Pak et al. 1991), and all members
of this gene family are alternatively spliced. Interestingly, there
has been no demonstration of isoform-dependent variability in
inactivation kinetics in heterologous expression systems (Baro
et al. 2001; Po et al. 2001). Significant heterogeneity in Shal
inactivation kinetics measured in situ, however, has been observed in cultured embryonic Drosophila neurons (Tsunoda
and Salkoff 1995) and has also been seen in the lobster stomatogastric ganglion (Golowasch et al. 1999). In Drosophila,
the shift between kinetic forms was very fast and seen at the
single channel level, leading the authors to postulate phosphorylation as a mechanism of Shal modulation. Auxiliary subunitdependent changes in the voltage dependence of inactivation
have been demonstrated for Kv4.3 and for lobster Shal (Decher
et al. 2001; Zhang et al. 2003). Proteins of the KChip/Frequenin family were able to selectively modulate inactivation while
minimally affecting activation. In Drosophila, a mutant that
overexpresses Frequenin has alterations in IA (Poulain et al.
1994). Differences in the Shal currents in third instar type Ib
and Is motor neurons could be a consequence of differential
expression of a member of the KChip/Frequenin family.
J Neurophysiol • VOL
2363
Implications of Ib/Is differences for locomotor activity
The similarities in firing behavior between MNISN-Is and
MNSNb/d-Is and the similarities within the Ib neuron group
suggests the possibility that there are inherent functional and
molecular differences between these morphologically distinct
neurons. Type Is neurons innervate groups of longitudinal and
oblique muscles of a given hemisegment dorsally (MNISN-Is),
ventrally (MNSNb/d-Is), and laterally (MNSNa-Is), allowing
these neurons to control or modulate a large number of functionally related muscles in concert. This sharply contrasts with
role of the Ib neurons which innervate only one or two muscles, and may act as muscle-specific “drivers.” Activation of
(groups of) individual muscles by Ib neurons may provide a
fine tuning of locomotion.
The delayed spiking mediated by Shal has been observed in
other systems involving motor pattern generation. In Aplysia,
L14 motor neurons, which control inking, fire action potentials
only after a critical build-up of synaptic input that is able to
inactivate transient potassium currents (Byrne et al. 1979).
These neurons display strikingly similar spiking behavior to
the Is neuron under current clamp. Whereas a sudden suprathreshold input will produce a delayed, slow spiking activity, a
tonic subthreshold input could make instantaneous bursting
activity more likely on suprathreshold input. This sort of activity dependence is particularly pertinent to the activity driving locomotor muscles as the particular sequence of driving
input can affect contraction timing and amplitude. Also, the
insensitivity of the neuron to short, nonsummating input may
allow the motorneuron to act as a low-pass filter (Byrne et al.
1979). Further study of the differences between the two subtypes will elucidate how locomotor behavior is organized in the
larval system.
Direct study of CNS neurons in the Drosophila third
instar larva
The ventral ganglion preparation developed in this study has
allowed direct access to identified central neurons in Drosophila. Previous studies using cultured larval neurons have documented the great diversity of morphological and electrophysiological properties in such preparations (Zhao and Wu 1997).
A recent study by Rohrbough and Broadie in a similar ventral
ganglion preparation has already shown great promise of
this system as a means to study central synaptic transmission
(Rohrbough and Broadie 2002). The stereotyped connectivity
and functional properties of the motor neurons characterized in
this study will allow genetics and pharmacology to be used to
determine the molecular basis of modulation and cellular behavior. The function of specific motor neurons in multiply
innervated muscle fibers as well as their role in the network of
neurons effecting locomotor behavior can now be studied
within the intact larval system.
ACKNOWLEDGMENTS
We thank E. Marder for helpful discussions and E. Dougherty for help with
figures. We also thank J. Rohrbough for discussing details of his larval CNS
recording studies.
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2364
J. C. CHOI, D. PARK, AND L. C. GRIFFITH
GRANTS
This work was supported by National Institutes of Health Grants P01
GM-33205 and MH-067284 to L. C. Griffith and Training Grant P32 NS07292 to D. Park.
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