Seminario 2

Yue Dai, Kevin P. Carlin, Zongming Li, Douglas G. McMahon, Robert M.
Brownstone and Larry M. Jordan

J Neurophysiol 102:3365-3383, 2009. First published Sep 30, 2009; doi:10.1152/jn.00265.2009
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Multiple Effects of Serotonin and Acetylcholine on Hyperpolarization-Activated Inward
Current in Locomotor Activity-Related Neurons in Cfos-EGFP Mice
Y. Dai and L. M. Jordan
J Neurophysiol, July 1, 2010; 104 (1): 366-381.
[Abstract] [Full Text] [PDF]
Multiple Patterns and Components of Persistent Inward Current With Serotonergic

Modulation in Locomotor Activity-Related Neurons in Cfos-EGFP Mice
Y. Dai and L. M. Jordan
J Neurophysiol, April 1, 2010; 103 (4): 1712-1727.
[Abstract] [Full Text] [PDF]
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J Neurophysiol 102: 3365–3383, 2009.
First published September 30, 2009; doi:10.1152/jn.00265.2009.
Electrophysiological and Pharmacological Properties of Locomotor

Activity-Related Neurons in cfos-EGFP Mice
Yue Dai,1 Kevin P. Carlin,1 Zongming Li,2 Douglas G. McMahon,4 Robert M. Brownstone,2,3
and Larry M. Jordan1
1
Department of Physiology; University of Manitoba, Winnipeg, Manitoba, Canada; 2Departments of Anatomy and Neurobiology
and 3Surgery (Neurosurgery), Dalhousie University, Halifax, Nova Scotia, Canada; and 4Department of Biological Sciences, Vanderbilt
University, Nashville, Tennessee
Submitted 25 March 2009; accepted in final form 25 September 2009
Dai Y, Carlin KP, Li Z, McMahon DG, Brownstone RM, Jordan LM. Kudo 2000; Nishimaru et al. 2000; Palmer et al. 2001). Early
Electrophysiological and pharmacological properties of locomotor activity- efforts to examine the properties of rodent neurons active
related neurons in cfos-EGFP mice. J Neurophysiol 102: 3365–3383, 2009. during locomotion included intracellular and blind patch re-
First published September 30, 2009; doi:10.1152/jn.00265.2009. Although
locomotion is known to be generated by networks of spinal neurons, cordings (Kiehn et al. 1996; MacLean et al. 1995), tetrode

knowledge of the properties of these neurons is limited. Using recordings (Tresch and Kiehn 1999), and calcium imaging
neonatal transgenic mice that express enhanced green fluorescent (Nakayama et al. 2002) from the isolated neonatal rat spinal
protein (EGFP) driven by the c-fos promoter, we visualized EGFP- cord.
positive neurons in spinal cord slices from animals that were subjected Recently techniques have become available to identify spe-
to a locomotor task or drug cocktail [N-methyl-D-aspartate, serotonin cific interneurons in reduced spinal cord preparations such that
(5-HT), dopamine, and acetylcholine (ACh)]. The activity-dependent
their physiological and pharmacological properties can be
expression of EGFP was also induced in dorsal root ganglion neurons
with electrical stimulation of the neurons. Following 60 –90 min of examined. For example, it has been shown that the activity of
swimming, whole cell patch-clamp recordings were made from ascending commissural cells can be modulated by the neuro-
EGFP⫹ neurons in laminae VII, VIII, and X from slices of segments transmitters serotonin (5-hydroxytryptamine, 5-HT) and ace-
T12 to L4. The EGFP⫹ neurons (n ⫽ 55) could be classified into three tylcholine (ACh) (Carlin et al. 2006; Zhong et al. 2006a).
types based on their responses to depolarizing step currents: single Similarly, descending commissural cells are excited by 5-HT
spike, phasic firing, and tonic firing. Membrane properties observed in and are rhythmically active during fictive locomotion in vitro
these neurons include hyperpolarization-activated inward currents (Zhong et al. 2006b). Populations of ventral interneurons,
(29/55), postinhibitory rebound (11/55), and persistent-inward cur- including EphA4 (Butt et al. 2005; Kullander et al. 2003),
rents (31/55). Bath application of 10 – 40 ␮M 5-HT and/or ACh
Dbx1 (Lanuza et al. 2004), En1 (Gosgnach et al. 2006), Chx10
increased neuronal excitability or output with hyperpolarization of
voltage threshold and changes in membrane potential. 5-HT also (Crone et al. 2008), and Sim (Zhang et al. 2008) expressing
increased input resistance, reduced the afterhyperpolarization (AHP), neurons have been shown to have discrete roles in locomotion.
and induced membrane oscillations, whereas ACh reduced the input HB9-expressing interneurons may also play a role in rhythm
resistance and increased the AHP. In this study, we demonstrate a new generation (Brownstone and Wilson 2008; Hinckley et al.
way of identifying neurons active in locomotion. Our results suggest 2005; Wilson et al. 2005). A discrete group of cholinergic
that the EGFP⫹ neurons are a heterogeneous population of interneu- medial partition neurons located near the central canal appears
rons. The actions of 5-HT and ACh on these neurons provide insights to terminate on motoneurons and control their excitability
into the neuronal properties modulated by these transmitters for during locomotor activity by reducing the action potential
generation of locomotion. hyperpolarization (Miles et al. 2007). Some of these geneti-
cally defined neurons can now be visualized in spinal cord
slices through the use of fluorescent reporter genes. Neverthe-
INTRODUCTION
less, few of these neurons have been extensively characterized
It is well established that the basic rhythm and pattern of in terms of their electrophysiological properties and their
locomotion is generated by a system of spinal neurons that response to neurotransmitters that produce fictive locomotion
make up the central pattern generator (CPG) for locomotion for instance.
(Brown 1911; Grillner 1981). Numerous studies have been These efforts to define locomotor neurons in the spinal cord
undertaken to identify neurons that make up the CPG. It was differ from the present study by virtue of the fact that they
initially suggests that the rat CPG is anatomically localized to employed anatomical and genetic features for identification of
the L1/L2 spinal segments (Cazalets et al. 1995, 1996), but the neurons. In contrast we use a marker of neural activity—
subsequent studies suggest that the hindlimb network is dis- enhanced green fluorescent protein (EGFP) expression driven
tributed throughout the lumbar and supralumbar spinal cord by the c-fos promoter—to identify neurons active during a
(Cina and Hochman 2000; Cowley and Schmidt 1997; Kjaer- locomotor task in mice. In a recent study (Dai et al. 2005b), we
ulff et al. 1994; Kremer and Lev-Tov 1997; Nishimaru and have demonstrated that locomotion induces cfos expression in
decerebrate cat spinal neurons. Here we use a degradable form
Address for reprint requests and other correspondence: L. M. Jordan, Dept. of EGFP to minimize any labeling from activity prior to a
of Physiology, University of Manitoba, Winnipeg, Manitoba R3E 3J7, Canada locomotor task and then target these neurons for study in spinal
(E-mail: larry@scrc.umanitoba.ca). cord slices. We characterize the electrophysiological properties
www.jn.org 0022-3077/09 $8.00 Copyright © 2009 The American Physiological Society 3365
3366 DAI, CARLIN, LI, McMAHON, BROWNSTONE, AND JORDAN
of EGFP-positive neurons (“locomotor neurons”) in lamina was removed from the spinal column, fine forceps were used to pull
VII, VIII, and X of T12–L4 segments using whole cell patch- the ganglia from between the vertebrae. This first part of the procedure
clamp techniques. Previous studies demonstrated that 5-HT is was performed in ice-cold dissecting artificial cerebrospinal fluid
necessary and ACh is sufficient to produce locomotor-like (ACSF). Once removed from the animal, the ganglia were placed in a
conical tube containing room-temperature HEPES-based ACSF solu-
activity in isolated spinal cords of neonatal rats and mice tion with 2.5% trypsin (Invitrogen, Carlsbad, CA). The trypsin was
(Cowley and Schmidt 1994; Hochman et al. 1994a; Jiang et al. allowed 20 min to work before the digested tissue was rinsed (⫻3)
1999; Kiehn and Kjaerulff 1996; MacLean et al. 1995; Smith with fresh HEPES ACSF containing bovine serum albumin (Sigma).
and Feldman 1986, 1987; Smith et al. 1988). Further studies The tissue was then triturated with a plastic pipette and plated directly
show that several types of 5-HT receptors (5-HT1, 5-HT2, and into the glass-bottomed recording chamber. HEPES-based ACSF
5-HT7) are essential in inducing locomotor-like movements in contents were (in mM) 140 NaCl, 3 KCl, 10 HEPES, 2 MgCl2, 10
neonatal rat and mouse spinal cord (Beato and Nistri 1998; glucose, and 2 CaCl2, pH ⫽ 7.4.
Bracci et al. 1998; Cazalets et al. 1992, 1995; Hochman et al.
2001; Lui and Jordan 2005; Madriaga et al. 2004) and in adult Imaging of acutely dissociated DRG
rat and mouse transected spinal cord (Landry and Guertin
2004; Landry et al. 2006; Ung et al. 2008). We therefore Fluorescent images were captured using a Hamamatsu image cap-
ture and processing package (Hamamatsu, Bridgewater, NJ). This
examined the modulation of intrinsic membrane properties of included a C2400 CCD camera, a camera controller, ARGUS 20
the EGFP⫹ neurons by these two neuromodulators. image processor, and HPS frame grabber and software. The images
We demonstrate that activation of neurons from the cfos- were acquired at regular intervals of 1–2 min. Frames (512) were
EGFP mice results in robust expression of EGFP sufficient for accumulated with the ARGUS software, and images were stored on a

identification of these neurons and report heterogeneous ana- Pentium class computer. The fluorescent light shutter was closed
tomical and electrophysiological properties and responses to when not acquiring images. Using control cells alone or unstimulated
5-HT and ACh. Preliminary reports of some of this work have cells in the field of view (n ⫽ 4; see RESULTS) the illumination process
been published (Brownstone et al. 2002; Dai et al. 2004). itself did not cause any detectable increase in fluorescence expression.
This is in contrast to the use of a long-pass filter (XF-02; Omega
Optical), which caused an increase in GFP expression in proportion to
METHODS the rate of illumination (data not shown). This latter increase in
The animal protocol was approved by the Dalhousie University fluorescence signal is likely due to UV stimulated c-fos expression.
Animal Care Committee, the University of Manitoba Central Animal Intensity measurements were preformed on stored images with the
Care Services and conformed to the standards of the Canadian ARGUS software. Intensity difference was calculated using defined
Council on Animal Care. areas in the cell of interest and a reference cell or background.
Production of the cfos-EGFP transgenic mice Preparation of slices and patch-clamp recordings
Experiments were carried out on neonatal (P4-15) cfos-EGFP mice.
C-FOS PROMOTER-PD2EGFP-1. The cfos-EGFP mouse line was pro- A locomotor task (walking or swimming) was induced in the animals
duced in the McMahon lab in the following manner: plasmid p301- prior to preparation of slices. The walking protocol was used for only
602 (Gilman et al. 1986) was cut with HindIII and ligated to an EcoRI older mice (⬎P8). With this protocol, a small amount of mother
adaptor. The plasmid was then cut with BamHI to release a 700-bp mouse’s fur was used to attract the animals. The fur was put into a
EcoRI/HindIII-BamHI fragment that encompassed the mouse c-fos microcentrifuge chamber. The chamber was then placed near the nose
promoter (Accession No. V00727, nucleotides 1– 600 plus about 100 of the animals and moved slowly to attract the animals for walking.
unsequenced base pairs upstream), including the following response The swimming protocol was designed for younger mice of P4 –P8.
elements: CRE, AP1, SIE, SRE. The gel-purified EcoRI-HindIII- Animals at this age were usually tiny in size and physically weak for
BamHI fragment was cut with EcoRI and ligated into EcoRI-BamHI long walking (60 –90 min) with body weight. Therefore this protocol
cut pd2EGFP-1 vector. DH5⬘ alpha subcloning efficiency (Gibco) was used to induce locomotor task in ⬎90% of the animals (n ⫽ 64:
cells were transfected with the c-fos promoter-GFP plasmid following 44 for patch-clamp experiments and 20 for confocal images) in this
manufacturer’s instructions (calcium phosphate). The c-fos promoter- study. A length of 1-cm-wide paper tape is prepared for suspending
GFP plasmid was purified (InVitrogen, SNAP Midi Prep), cut with the animal by making the portion of tape that touches the animal
EcoRI and AflII, releasing a 1.7-kb base-pair fragment that was nonadhesive with a 4-cm-long piece of the same tape placed sticky
subsequently gel purified. Twenty micrograms of EcoRI-AfIII frag- side to sticky side. This tape is then placed around the animal’s thorax,
ment was purified by cesium chloride gradient without ethidium and the distal ends are joined over the animal’s back, leaving ample
bromide and then extensively dialyzed against TEN buffer (1 l, 6 length of tape to allow the attachment of a clamp for suspension of the
times) at 4°C. animal over a beaker of water. The tape allows the mouse full range of
Transgenic mice were generated by the University of Kentucky motion in all limbs as well as normal breathing. The ends of the tape are
Transgenic Mouse Facility. Transgene DNA was microinjected (5 placed in a clamp attached to a calibrated microdrive. The water bath
ng/␮l) into the pronuclei of fertilized B6C3F1 hybrid mouse embryos is held at 26 –30°C (usually 28°) using a hotplate and thermometer.
(Harlan) and implanted into pseudopregnant females. Mice were The microdrive is used to lower the mouse into the water bath. The
screened for the transgene both by dot-blot analysis and PCR of clamp is situated on the tape in such a manner as to assure that the
genomic DNA isolated from the tail at the time of weaning. A head is maintained above the surface of the water at all times. On
homozygous transgenic line was then bred. being positioned with its limbs in the water, the mouse immediately
begins swimming. During the first 5–10 min, the mouse is allowed to
Dorsal root ganglion neuron (DRG) dissociation habituate to the water bath. During this time, the animal may attempt
to escape the water. If this occurs, the experimenter lifts the animal
DRG were harvested from postnatal (P4 –P13) mice. Harvesting of out of the water for a few seconds and then places it back in. This is
these cells used the same surgical procedure as described previously repeated a few times until the mouse becomes used to the water and
to harvest the spinal cord (Carlin et al. 2000). Once the spinal cord ceases all signs of struggling. At this point, the animal begins to swim
J Neurophysiol • VOL 102 • DECEMBER 2009 • www.jn.org

LOCOMOTOR-ACTIVATED NEURONS IN cfos-EGFP MICE 3367
steadily for as long as its limbs are kept below the surface of the water. mined by step currents with 0.5-s duration and 50 pA for each step.
This “swimming task” continues for 60 –90 min. Maintaining the The minimum step that could evoke a single spike or repetitive firing
temperature within the proper range is a key element to the success of was taken as the Ith. The Vth was defined as the membrane potential at
the task. If the water bath is too warm, the mouse may fall asleep. If which the rising rate of dV/dt ⬎10 mV/ms. The Vth reported in this
it is too cold, the animal will struggle. The duration of the task must paper was measured from the first spike of firings evoked by a 2-s
be ⱖ60 min to assure that the locomotor activity is sufficient to induce triangular current with amplitude of 0.5–1.5 nA. The resting mem-
c-fos expression in the locomotor neurons of the spinal cord and brane potential was monitored throughout the recordings using Axon
consequent expression of the EGFP protein in these neurons. The Minidigi 1A. The reported Em in this paper was calculated from the
EGFP allows the locomotor neurons activated by the task to be membrane potentials averaged over 100 ms prior to the step currents
visualized later in spinal cord slices taken from this mouse. The reason used for determining the Ith. The measurements of AP and AHP
for using swimming task is that this new task is much more efficient properties were based on an averaged spike from the first one to five
in inducing locomotion-like movement in young animals than the spikes evoked by Ith. The Vth was used as reference value to measure
walking task. No overt difference in expression of EGFP⫹ neurons the AP height and width and AHP depth. The half-decay time of AHP
was found between the walking and swimming slices. was measured from the time of AHP peak to the time of half AHP
The procedure for the preparation of spinal cord slices was as decaying from the peak. Step currents with duration of 2 s and step of
previously reported (Carlin et al. 2000). Transverse slices were cut at 50 pA were injected into the cells to determine the F-I relations. The
thickness of 180 –250 ␮m from T12 to L4 segments and remained in instantaneous firing frequency was calculated as the reciprocal of the
the recovering ACSF for ⱖ1 h before the patch-clamp recordings. The interspike interval (ISI) for the first, second, and steady-state (ss)
slices were then transferred to a recording chamber mounted in the spikes. The ss firing frequency was calculated as a mean rate of firings
stage of an upright Olympus BX50 microscope fitted with differential over the last 1 s duration of the firings in the tonic firing neurons (type
interference contrast (DIC) optics and epifluorescence. The chamber 3) or over the last 5–10 spikes in the neurons with phasic firing (type

was perfused with recording ACSF at rate of 2 ml/min, bubbled with 2). In a few of the type 2 cells that fired only a few spikes, the
95% O2-5%CO2. The EGFP-positive neurons were identified at ⫻40 averaged rate of firing for all spikes was taken as the ss firing
magnification using epifluorescence with a narrow band GFP cube frequency. A linear regression was applied to the F-I relations for
(Omega XF-37). The neurons were then visualized using infrared calculation of the slopes. The slopes calculated from the F-I relations
illumination and images collected (Hamamatsu camera controller of the first, second, and ss spikes were defined as slope 1, slope 2, and
C2400 and ARGUS image processor). The visualized neurons were slope SS, respectively.
patched (electrode resistances: 3– 6 M⍀). A MultiClamp 700A patch- A 1-s hyperpolarizing step current with step of ⫺20 to ⫺50 pA was
clamp amplifier, Digidata 1322A A/D converter, Minidigi 1A, and injected into the cells. The voltage deflection with no or minimum
pClamp (9.0) software (all from Molecular Devices) were used for activation of h-current was averaged over the first 0.5 s starting from
data acquisition. Whole cell patch recordings were made in current- the peak deflection of the membrane potential. The Rin was calculated
clamp mode with bridge balance and capacitance compensation and in by the mean value of the voltage divided by the amplitude of the
voltage-clamp mode with series resistance compensation by 70 – 80%. corresponding step current. The time constants (␶m and ␶1) were
A constant hyperpolarizing current from 0 to 100 pA was injected into determined by fitting a double decaying exponential function with
some of the cells to maintain the resting membrane potential at about form of Y ⫽ y0 ⫹ A1 ⴱ Exp (V/␶m) ⫹ A2 ⴱ Exp(V/␶1) to the averaged
⫺60 mV or lower during the recordings. The recording protocols were voltage responses to a train of five pulses (0.5 ms, ⫺1 nA, 250-ms
repeated two to three times in each condition (control, drugs, and interval). The whole cell capacitance was calculated by the formula:
washout etc.) with ⬃30 s apart between two successive recordings. Cm ⫽ {␶m ⴱ L/[Rin ⴱ Tanh(L)]}, where L ⫽ ␲/(␶m/ ␶1 ⫺ 1)1/2.
Normally recordings were made in 2– 8 min following drug applica- In addition to the measurement of the preceding membrane prop-
tion and repeated for ⬃10 min before switching to new conditions. To erties, persistent inward current (PIC) and hyperpolarization activa-
have the recordings from healthy cells, the time for intracellular tion inward current (Ih) were also tested in the present study. The PIC
recording was usually ⬍40 min. Data were low-pass filtered at 3 kHz was recorded in voltage clamp by applying a slow voltage bi-ramp
and sampled at 10 kHz. The data were analyzed using self-written with duration of 5–10 s, peak of 0 –30 mV, and holding potential of
codes with Igor Pro (4.0) and Axon Clampfit (9.0). Student’s t-test and ⫺70 mV (Lee and Heckman 2001). Ih was recorded in current clamp
ANOVA were performed with statistical significance defined as P ⬍ with the same protocol for Rin recording.
0.05. Results are shown as means ⫾ SD.
Solutions and chemicals
Confocal images The dissecting ACSF contained (in mM) 25 NaCl, 188 sucrose, 1.9
Slices of both control and locomotion mice were cut at 100 –150 KCl, 1.2 NaH2PO4, 10 MgSO4, 26 NaHCO3, 1.5 kynurenic acid, and
␮m and fixed for 20 min with 4% paraformaldehyde. They were then 25 glucose. The recovering ACSF contained (in mM) 118 NaCl, 4.5
mounted on slides, allowed to air dry, and fitted with coverslips with KCl, 1.2 NaH2PO4, 10 MgSO4, 26 NaHCO3, 1.0 CaCl2, 1.5 kynurenic
Vectashield mounting medium (Vector Laboratories H-1000). Slides acid, and 25 glucose. The recording ACSF contained (in mM) 130
were scanned and photographed with step of 1.5 ␮m and thickness of NaCl, 4.5 KCl, 1.25 NaH2PO4, 1.25 MgCl2, 26 NaHCO3, 2.5 CaCl2,
15–50 ␮m using confocal laser scanning microscope (Olympus Fluo- and 10 glucose. The pH of these solutions was ⬃7.4 when bubbled
view 2.1). The thickness of slices for scanning was chosen appropri- with 95% O2-5%CO2.
ately to avoid image saturation while the clear distribution of EGFP- The intracellular solution contained (in mM) 150 KMeSO4, 10
positive cells was preserved. NaCl, 10 HEPES, 0.1 EGTA, 3 Mg-ATP, and 0.3 GTP. Alexa 594
(⬃3%; Invitrogen Canada) or Lucifer yellow (⬃1%) was added to the
intracellular solution in some experiments to study the morphology of
Measurement of cell membrane properties the recorded neurons. All chemicals were purchased from Sigma (St.
The membrane properties measured and calculated in this study Louis, MO) unless otherwise noted.
include current threshold (Ith), voltage threshold (Vth), resting mem-
brane potential (Em), input resistance (Rin), membrane time constant RESULTS
(␶m and ␶1), whole cell capacitance (Cm), slope of frequency/current
(F-I) relation, action potential (AP) height and width, and afterhyper- The present study is based on the experimental results from
polarization (AHP) depth, and half-decay time. The Ith was deter- intracellular recordings of 72 spinal neurons (60 EGFP⫹ and
12 EGFP⫺) from 44 cfos-EGFP mice of P4-15; data of 6 DRG EGFP expression in spinal cord slices in vitro
cells from P4 –P13 cfos-EGFP mice; and confocal images from
20 cfos-EGFP mice of P4-15. The details of the results are Previous studies have shown that certain neurotransmitters
presented in the following text. applied directly to the isolated spinal cord of the neonatal rat or
mouse can induce locomotor-like activity (see Kiehn and Butt
2003 for review). To determine whether EGFP expression
EGFP expression in acutely dissociated DRG cells could be evoked in vitro by these same agents in slices from the
cfos-EGFP transgenic mice, we applied a mixture of 30 ␮M
The activity-dependent nature of the c-fos-driven EGFP 5-HT, NMDA, dopamine, and ACh to slices of 150 ␮m
expression was confirmed using acutely dissociated dorsal root thickness taken from the T12–-L4 segments of P8 cfos-EGFP
ganglion neurons. These neurons were chosen because of their transgenic mice. After 2 h, control and drug-treated slices were
high degree of viability during the dissociation process as well fixed and examined using a laser scanning confocal micro-
as a previous report of c-fos expression in these cells due to scope. The number and intensity of EGFP⫹ cells were dra-
trains of action potentials (Fields and Nelson 1994). Once matically increased in the drug-treated slices. The intensity of
dissociated, these cells were patch clamped and repeatedly the EGFP⫹ neurons was increased by the drug cocktail to a
depolarized under voltage-clamp conditions (Vh ⫽ ⫺60 mV, degree that necessitated that the sensitivity (gain) of the camera
test pulse to 0 mV ⫻ 500 ms ⫻ 0.2 Hz) while fluorescence had to be reduced by 20 –30% of control to avoid saturation of
intensity measurements were taken at regular intervals. Using the images. As Fig. 2 shows qualitatively, the drug cocktail
this procedure, stimulated cells were seen to fluoresce more induced a dramatic increase in the EGFP detectable in neurons
intensely during stimulation (n ⫽ 5/6). In a number of exper- in most laminae of the spinal cord. A pronounced increase in

iments, multiple cells were included in the field of view, and EGFP expression occurred in the ventral horn, in lamina VII,
the difference of intensities was recorded for stimulated and VIII, and IX, and around the central canal (lamina X). Mo-
unstimulated cells. As illustrated in Fig. 1 (A–D), the fluores- toneurons as well as interneurons displayed increased fluores-
cence intensity of the stimulated cell can be seen to increase cence, showing that many cells in the presumed locomotor
dramatically, while it is unchanged in the unstimulated cell. areas of the spinal cord including the ventromedial region (see
The accompanying graph (Fig. 1E) demonstrates that the Kiehn and Kjaerulff 1998; Kjaerulff and Kiehn 1996) are
fluorescence increase corresponds to the period of stimulation, activated during the chemical stimulus and that these cells can
and after a short delay, the intensity decreases with cessation of be detected with the EGFP reporter. These experiments dem-
the stimulation. The rapidity of these fluorescence changes is onstrate that the c-fos-EGFP mouse model can be used to
consistent with the expression of a fos-␤-galactosidase fusion reveal neurons responsive to bath application of drugs in slice
protein that showed increased expression in as little as 15 min preparations.
poststimulation in transfected neuroblastoma cells (Schilling et
al. 1991). The decay of fluorescence intensity was slow and In vivo locomotor activity induces EGFP expression
could continue for hours before returning to baseline. These
experiments demonstrate that neurons from the c-fos-EGFP To determine whether these animals could be used to study
animals used in this study express EGFP under conditions that neurons activated following locomotion, locomotor activity
should increase the expression of c-fos. (swimming) was induced in intact cfos-EGFP transgenic mice
A B C D FIG. 1. Enhanced green fluorescent pro-

tein (EGFP) expression in acutely dissoci-
DIC 6 min 32 min 62 min
ated dorsal root ganglion cells. Dissociated
cells were patch clamped and repeatedly de-
polarized under voltage-clamp conditions
while measurements of fluorescence inten-
sity difference were taken at regular inter-
vals. A: multiple cells were included in the
field of view, and the difference of intensities
were recorded for stimulated and unstimu-
lated cells. B: the fluorescence intensity of
the stimulated cell was increased after 6-min
stimulation while it was unchanged in the
E 2500 unstimulated cell. C: the fluorescence inten-
C
sity of the stimulated cell reached a peak
2000 even after termination of the stimulation.
After a short delay, the intensity decreased.
Intensity difference
1500 D
D: the decay of fluorescence intensity ap-
pears to be a biphasic decay process, an
1000
initial fast phase and a 2nd slower phase. The
500 2nd phase could take hours to return to
B
baseline. E: plot of fluorescence intensity
0 difference over stimulation time. The stimu-
lation started at time 0 and terminated at time
-500 A 24 min (- - -). Three time intervals (B–D)
Time (min)
0 10 20 30 40 50 60 70
were chosen to show the changes in fluores-
cence intensity in B–D, respectively.
stimulation on stimulation off

Control 5HT, NMDA, Dopamine and ACh
Dorsal Dorsal
FIG. 2. EGFP expression in spinal cord slices in vitro. A

cocktail of 30 ␮M serotonin (5-HT), N-methyl-D-aspartate
(NMDA), dopamine, and ACh was applied to slices of 150
␮m thickness taken from the T12–L4 segments of P8 cfos-
EGFP transgenic mice. After 2 h, control (left) and drug-
treated slices (right) were fixed and examined using a laser
scanning confocal microscope. The drug cocktail induced a
dramatic increase in the EGFP detectable in neurons in most
laminae of the spinal cord. A pronounced increase in EGFP
expression occurred in the ventral horn, in lamina VII, VIII,
and IX, and around the central canal (lamina X).

100 µm
P8 Ventral P8 Ventral
for a period of 60 –90 min. Slices prepared and fixed from these An example of an EGFP-positive neuron patched in the
animals were compared with slices from control littermates. target area is shown in Fig. 4A. The fluorescence image of
Figure 3 shows confocal images from control (left) and loco- EGFP could be visualized on the monitor (Fig. 4A1). The same
motion (right) groups from P4 (top), P6 (middle), and P8 cell is shown in an infrared image (Fig. 4A2, arrow) and
(bottom) mice. In the ventral horn, numerous cells were labeled patched with a glass pipette electrode (A3). This cell was
with EGFP in lamina VII, VIII, and X in the locomotion group, located in lamina VIII as shown in a low-power (⫻10) infrared
whereas only a few cells were labeled in the same region in the image (Fig. 4A4). Figure 4B shows an EGFP⫹ neuron filled
control group. Thus it is clear that the swimming task induced with Lucifer yellow after intracellular recordings. The image
EGFP expression in neurons in the locomotor area of the displays the detailed morphology of the neuron (round soma
ventral horn. In this study, lamina VII, VIII and X were set as with ⬎4 stem dendrites). Figure 4C is an illustration of the
target areas from which the EGFP⫹ neurons were selected for laminar distribution of 39 EGFP⫹ neurons patched in the target
patch-clamp recordings. area for which the low-power images of the electrode placement
EGFP-positive cells were also seen in dorsal horn areas in were available. Cells located in the left side of the slice were
both control and locomotion groups. This is not surprising, as mirrored onto right side.
dorsal horn neurons are expected to be activated and express
Fos during both the locomotor task (Dai et al. 2005b) and the Three types of EGFP⫹ neurons
surgical procedures themselves (Coggeshall 2005). Neurons in
the dorsal horn were not targeted in this study, but it is striking The EGFP⫹ neurons in lamina VII, VIII, and X could be
that a group of neurons in the medial part of the dorsal horn classified into three types based on their firing patterns in
(laminae I–VI) was activated due to the locomotor task. Recent response to depolarizing step currents: single spike (type 1),
observations demonstrate rhythmically active neurons in this phasic firing (type 2), and tonic firing (type 3). The similar
region during locomotor activity (J. M. Wilson, E. Blagov- firing pattern has been reported in rat spinal neurons in ventral
echtchenski, and R. M. Brownstone, unpublished data). Figure horn (Szucs et al. 2003; Theiss and Heckman 2005; Theiss et
3 also shows that the EGFP-labeled cells are reduced with age al. 2007), superficial horn (Prescott and De Koninck 2002), and
in both control and locomotion groups. However, the labeled deep dorsal horn (Hochman et al. 1997). In response to a
cells appear constantly with less correlation to the animal ages depolarizing step current, neurons of type 1 elicited only one to
compared with the control ones. The dramatic increase in two spikes (Fig. 5A1); the type 2 fired briefly with firing duration
EGFP-labeled cells in locomotion groups validates this animal ⬍1 s (B1); the type 3 fired repetitively for a few or tens of seconds
model for the present study. (C1). In addition to differences in firing patterns, the three types of
EGFP⫹ neurons also showed differences in some electrophysio-
logical properties, such as current threshold and input resistance
Electrophysiological properties of EGFP-positive neurons in
(Fig. 5). In Fig. 5A1 a single spike was elicited by a step current
the ventral horn
of 100 pA for 2-s duration in a type 1 cell in lamina VII. The Rin
The results presented in this study were based on the data was calculated as 230 M⍀ for this cell (Fig. 5A2). With half the
collected from 55 neurons sampled in laminae VII, VIII, and amount of the step current (50 pA), brief firing was evoked in a
X. Of the 55 neurons, 43 neurons were EGFP positive and 12 type 2 cell in lamina X (Fig. 5B1), which had a Rin of 457 M⍀
were non-EGFP positive. The 55 neurons were selected for (Fig. 5B2). The same amount of current produced tonic firing in a
data analysis based on the criteria that resting Em ⬇ (or ⬎) type 3 cell in lamina VIII (Fig. 5C1). The Rin was 303 M⍀ for this
⫺60 mV, AP overshoot ⬎ (or ⬇) 0 mV, and Rin ⬎ 150 M⍀. cell (Fig. 5C2).
Control Locomotion
Dorsal Dorsal
200 m P4
P4 Ventral Ventral
FIG. 3. Target areas for EGFP⫹ neurons
for patch-clamp recordings. Locomotor activ-
ity was induced in GFP mice at age of P4 – 8.
Slices were cut from spinal cords T12–L4. Pho-
tos were taken at ⫻10 magnification using

Olympus confocal laser scanning microscope
(see METHODS for details). Left: control; right:
locomotion. Top: slices from P4 mice; middle:
from P6; bottom: from P8. Control slices show
that most of the EGFP-labeled cells are located
in dorsal horn areas from laminas I–VI. A few
of cells are also labeled near central canal area
(laminar X). These EGFP-labeled cells are re-
duced with animal age. The locomotion slices
show that in addition to the cells labeled in
dorsal horn areas, a lot of cells are labeled in
laminas VII, VIII and X. These labeled cells
appear constantly with less correlation to the
P6 P6 animal ages compared with the control ones.
The EGFP-positive cells in laminas VII, VIII,
and X are targeted for patch-clamp recordings.
P8 P8
Two subtypes of EGFP⫹ neurons were observed in our exper- highest Rin and lowest C, whereas the cells in lamina VII had the
iments based on their firing properties. One displayed a “slow” lowest Rin and highest Cm. These results may suggest a graded
type of repetitive firing (Szucs et al. 2003) and the other a distribution of the increased size in EGFP⫹ neurons from central
“delayed” type of phasic firing (Hochman et al. 1997; Prescott and cannel (lamina X) to ventral areas (lamina VII and VIII). The
De Koninck 2002). For simplicity, we classified the slow type majority of EGFP⫹ neurons in lamina VII were type 1 or type 3,
(n ⫽ 3) as type 3 and delayed type (n ⫽ 2) as type 2. Table 1A whereas lamina VIII neurons tended to be type 3. Lamina X
summarizes the membrane properties of 55 neurons (43 EGFP⫹ neurons were of all three types. Thus the EGFP⫹ neurons located
and 12 EGFP⫺) sorted in laminae VII, VIII, and X. No significant in the targeted laminae did not differ dramatically in terms of
difference is found between the EGFP⫹ and EFGP⫺ neurons in membrane properties or types, but the differences in Rin and
the membrane properties shown in the table. Laminar distributions Cm suggest that a detailed analysis of cell morphology may
of these neurons predicted differences only in input resistance and yield new ways of classifying locomotor neurons in the
whole cell capacitance. The neurons in lamina X displayed the ventral horn.
A EGFP+ neuron with patch clamp recording
A1 40X A2 40X
20 m
A3 40X A4 10X
FIG. 4. EGPF⫹ neurons with patch-clamp
recording. A1: an EGFP-positive neuron was

seen from a ⫻40 water-immersion lens with
EGFP fluorescence cube. A2: the same cell
(marked with white arrow) was observed in
infrared image. A3: the cell was patched with a
glass pipette electrode. A4: the same cell was
shown in a low-power (⫻10) lens. The elec-
trode position indicated the location of the cell
in laminar VIII. B: an EGFP⫹ neuron was
filled with lucifer yellow. The image shows the
80 m detailed morphology of the neuron: round
soma with ⬎4 stem dendrites. C: a laminar
Ventral distribution of 39 EGFP⫹ neurons patched for
recordings (shown as asterisk) was shown in
the target area lamina VII, VIII, and X.
B EGFP+ neuron filled with lucifer yellow C EGFP+ neurons (n=39) patched in target areas
Dorsal
V-VI
X
*
*
VII *** **** * * *
*
*** ** ** * *
IX ** * * * **
* **
* * * ** *
20 m VIII
40 m Ventral
IX
*
We also sorted the data according to the cell types to show had the minimum AP width and the maximum AHP height and
the difference in membrane properties. Because there is no half decay time.
significant difference between the EGFP⫹ and EFGP⫺ neu- The difference in firing patterns did not strictly correspond
rons, we put both groups together. The results are summarized to the difference in the neuron’s laminar location and mem-
in Table 1B. In general, type 1 neurons tended to possess the brane properties. The type 2 neurons were more concentrated
smallest Rin and AHP and the biggest Ith and Cm, whereas type in lamina X (73%, n ⫽ 15) than in other areas while neurons
2 neurons had the minimum Ith and Cm and the maximum Rin, of types 1 and 3 appeared evenly distributed in lamina VII,
␶m, and AP width. The type 2 neurons also showed a steeper VIII, and X. The functional significance of the condensed
slope of the F-I relation than that of type 3 neurons. The distribution of type 2 neurons is unknown. However, type 2
properties of type 3 neurons generally fell between those of neurons were significantly different from type 1 in Em, Ith, Rin,
types 1 and 2 expect for AP and AHP, where type 3 neurons and AP properties, suggesting that the type 2 neurons are a
A B C
A1 Single Spike B1 Phasic Firing C1 Tonic Firing
(Lamina VII) (Lamina X) (Lamina VIII)
20 mV
-70.2 mV -73.5 mV -60.4 mV
50 pA
0 pA
0.5 s
20 mV
A2 B2 C2
-69.3 mV -72.2 mV -61.7 mV
230 M
457 M 303 M
0 pA
-50 pA
0.5 s
FIG. 5. Three types of EGFP⫹ neurons. The EGFP⫹ neurons were classified into 3 types based on their firing patterns (A1–C1, top) in response to

depolarizing step currents (2 s, 50 pA for each step; A1–C1, bottom). The passive membrane responses (A2–C2, top) to hyperpolarizing step currents (1 s, ⫺50
pA for each step; A2–C2, bottom) were used to calculate input resistance by Ohm law. A: cells of single spike (type 1). A1: 2 step currents were injected into
a type 1 cell and only one single spike was elicited by the 2nd step (2 s, 100 pA). A2: a hyperpolarizing step current (1 s, ⫺50 pA) was injected into the same
cell, and the voltage deflection was used to calculate the Rin (230 M⍀). B: cells of phasic firing (type 2). B1: a 50-pA step current evoked a brief firing in a type
2 cell for ⬃0.6 s. B2: the cell membrane potential was hyperpolarized by a ⫺50-pA step current, and the Rin was calculated as 457 M⍀ in this cell. C: cells of
tonic firing (type 3). C1: the tonic firing in a type 3 cell was evoked by a 50 pA depolarizing step current. C2: the hyperpolarization of membrane potential was
produced by a ⫺50-pA step current. Rin ⫽ 303 M⍀ in this cell.
population of interneurons anatomically, electrophysiologi- based on electrophysiological properties and location, at least
cally, and functionally distinct from type 1 neurons. In addi- three distinct types of neurons can be identified in the ventral
tion, type 2 neurons also showed a significant difference from horn by their expression of EGFP following a locomotor task.
type 3 in F-I relations, AP, and AHP properties. Some studies
in rat spinal dorsal horn neurons suggested that the type 2 and Membrane currents mediated by multiple channels
3 cells might be two extremes of one heterogeneous cell
population (Szucs et al. 2003). The present study does not rule Hyperpolarization-activated inward current (Ih) was tested in
out this possibility. However, because the type 2 and 3 neurons this study. An example is shown in Fig. 6A (and Fig. 5B2). The
tended to be similar in passive properties (Em, Rin, Cm, etc) but Ih was activated in a type 1 neuron in lamina X by a step
different in active properties (AP, AHP, F-I relations), and current of ⫺50 pA and 1-s duration (Fig. 6A1). Some neurons
laminar distribution, it seems likely that these two types of also exhibited postinhibitory rebound after termination of the
neurons are functionally different. Significant differences in Vth step current. Figure 6A2 shows that two hyperpolarizing step
and AHP were also observed between type 1 and 3 cells, currents with ⫺50 pA for each step were injected into a type 3
suggesting that these two types of neurons should be dynam- neuron in lamina VII. A small Ih was activated by the first step
ically as well as functionally different as well. In summary, current with a spike elicited by the postinhibitory rebound. The
TABLE 1A. Membrane properties of the EGFP⫹ neurons classified in laminar distribution
Lamina VII (19) Lamina VIII (13) Lamina X (23) Significance EGFP⫹ (43) EGFP⫺ (12) Total (55)
Em, mV ⫺65.5 ⫾ 6 ⫺69.9 ⫾ 6 ⫺69.7 ⫾ 8 NS ⫺68.6 ⫾ 8 ⫺72 ⫾ 12 ⫺69.3 ⫾ 9

Tth, pA 89.5 ⫾ 51 111.5 ⫾ 58 91.3 ⫾ 49 NS 98.7 ⫾ 56 87.5 ⫾ 31 96.4 ⫾ 52
Rin (M⍀) 283.5 ⫾ 124 321.6 ⫾ 141 480.6 ⫾ 252 * 378.3 ⫾ 217 356.8 ⫾ 171 374.9 ⫾ 207
␶m, ms 21.0 ⫾ 16 19.7 ⫾ 9 24.8 ⫾ 13 NS 20.2 ⫾ 12 28.7 ⫾ 13 22.0 ⫾ 13
␶1, ms 4.7 ⫾ 2 4.4 ⫾ 2 3.8 ⫾ 2 NS 4.1 ⫾ 2 4.6 ⫾ 2 4.2 ⫾ 2
Cm, pF 145.1 ⫾ 67 124.7 ⫾ 52 88.7 ⫾ 43 * 107.7 ⫾ 50 128.8 ⫾ 76 115.8 ⫾ 59
Vth, mV ⫺45.8 ⫾ 11 ⫺38.5 ⫾ 10 ⫺37.3 ⫾ 11 NS ⫺39.5 ⫾ 8 ⫺42.9 ⫾ 17 ⫺40.1 ⫾ 11
AP height, mV 49.8 ⫾ 14 42.6 ⫾ 11 49.1 ⫾ 11 NS 46.2 ⫾ 13 53.1 ⫾ 11 47.7 ⫾ 12
AP width, ms 3.6 ⫾ 1 3.2 ⫾ 1 4.2 ⫾ 1 NS 3.7 ⫾ 1 4.0 ⫾ 1 3.8 ⫾ 1
AHP depth, mV 13.4 ⫾ 6 17.3 ⫾ 8 14.2 ⫾ 6 NS 14.5 ⫾ 6 14.1 ⫾ 6 14.4 ⫾ 6
AHP 1/2 decay, ms 46.3 ⫾ 34 47.4 ⫾ 30 53.1 ⫾ 41 NS 49.4 ⫾ 33 47.2 ⫾ 46 48.9 ⫾ 36
Slope 1, Hz/nA 175.9 ⫾ 74 165.4 ⫾ 168 204.8 ⫾ 93 NS 189.4 ⫾ 118 126.1 ⫾ 47 179.7 ⫾ 112
Slope 2, Hz/nA 143.5 ⫾ 68 117.8 ⫾ 93 147.1 ⫾ 55 NS 141.5 ⫾ 72 101.2 ⫾ 33 135.3 ⫾ 70
Slope SS, Hz/nA 122.3 ⫾ 66 82.2 ⫾ 30 112.8 ⫾ 43 NS 109.8 ⫾ 52 98.8 ⫾ 28 107.4 ⫾ 50
Type 1 7 2 4 10 3 13
Type 2 2 2 11 9 6 15
Type 3 10 9 8 24 3 27
Parentheses enclose n values. Values are means ⫾ SD. EGFP, enhanced green fluorescent protein. *, significant difference with P ⬍ 0.005 performed by
single-factor ANOVA for the cells’ laminar distributions. NS: no significant difference.

TABLE 1B. Membrane properties of the EGFP⫹ neurons classified in cell types
Significance (Student’s t-test)
Type 1 (Single) Type 2 (Phasic) Type 3 (Tonic) Total Mean Types 1 vs. 2 Types 2 vs. 3 Types 1 vs. 3
n 13 15 27 55
Em, mV ⫺66.7 ⫾ 5.4 ⫺70.6 ⫾ 5.3 ⫺67.4 ⫾ 8.5 ⫺69.1 ⫾ 9 † NS NS
Ith, pA 115.4 ⫾ 59.1 84.6 ⫾ 31.5 88.9 ⫾ 56 95.5 ⫾ 52 † NS NS
Rin, M⍀ 301.2 ⫾ 109.9 436.2 ⫾ 195.7 372.9 ⫾ 233.5 374.9 ⫾ 207 † NS NS
␶m, ms 23.4 ⫾ 14.9 25.8 ⫾ 10.8 20.4 ⫾ 13.4 22.3 ⫾ 13 NS NS NS
␶1, ms 4.2 ⫾ 1.6 3.9 ⫾ 1.9 4.1 ⫾ 2.6 4.2 ⫾ 2.3 NS NS NS
Cm, pF 129.7 ⫾ 46 104.6 ⫾ 52 109.3 ⫾ 53 116.7 ⫾ 59 NS NS NS
Vth, mV ⫺43.1 ⫾ 6.1 ⫺39.0 ⫾ 11.5 ⫺38.3 ⫾ 8.9 ⫺40.2 ⫾ 11 NS NS †
AP height, mV 49.9 ⫾ 10.7 47.7 ⫾ 11.3 47.5 ⫾ 14.2 48.1 ⫾ 12.6 NS NS NS
AP width, ms 3.5 ⫾ 0.6 4.5 ⫾ 1.3 3.3 ⫾ 1.2 3.7 ⫾ 1 † * NS
AHP depth, mV 10.7 ⫾ 4.6 13.4 ⫾ 6.2 16.7 ⫾ 6.1 14.5 ⫾ 6 NS † *
AHP 1/2 decay, ms 30.7 ⫾ 22.2 46.6 ⫾ 39.4 55.5 ⫾ 30.4 49.3 ⫾ 36 NS NS *
Slope 1, Hz/nA NA 197.1 ⫾ 67.6 176.1 ⫾ 126.8 182.2 ⫾ 112 NA NS NA
Slope 2, Hz/nA NA 143.7 ⫾ 51.4 133.2 ⫾ 77.2 136.2 ⫾ 70 NA NS NA
Slope SS, Hz/nA NA 134 ⫾ 51.8 96.9 ⫾ 46.1 106.9 ⫾ 50 NA † NA
Lamina VII 7 2 10
Lamina VIII 2 2 9

Lamina X 4 11 8
†: significant difference with P ⬍ 0.05; *, significant difference with P ⬍ 0.005; NS, not significant difference; NA, not applicable.
second step current produced a large Ih with an earlier elicita- from P4 –P8 mice, PIC was demonstrated in 51% of the
tion of the rebound spike. Ih was investigated in 67 neurons, neurons (34/67), 56% of the EGFP⫹ neurons (31/55), and 25%
(55 EGFP⫹ and 12 EGFP⫺). Ih was observed in 52% of the of EGFP- neurons (3/12). The expression of PICs was not
total neurons (35/67), 53% of the EGFP⫹ neurons (29/55), and related to the lamina distribution of the neurons.
50% of the EGFP⫺ neurons (6/12). Eleven of the 29 EGFP⫹
neurons that displayed Ih exhibited the postinhibitory rebound. 5-HT-induced membrane potential oscillations
This accounted for 38% of the EGFP⫹ neurons displaying Ih
(11/29) and 20% of the all 55 EGFP⫹ neurons (11/55). No 5-HT-induced membrane potential oscillations were ob-
correlation was found between Ih expression and neuronal type served in 7 of 20 cells tested. Similar observations were
or laminar distribution. reported in recent studies in ascending commissural interneu-
Persistent inward currents (PICs) were also tested in this rons in the neonatal mouse (Carlin et al. 2006; Zhong et al.
study. The currents could be explored by a slow voltage ramp 2006a) and in previous studies in rat spinal interneurons in
(Lee and Heckman 2001) as shown in Fig. 6B1, where a 5-s lamina VII (MacLean et al. 1995) and X (Hochman et al.
triangular voltage ramp from ⫺70 to 0 mV was applied to a 1994a,b). Figure 7 shows a voltage oscillation in a type 3
type 3 EGFP⫹ neuron in lamina VIII. A small PIC (⬃50 pA) neuron in lamina X after bath application of 20 ␮M 5-HT. The
with negative current slope was demonstrated on the rising 5-HT depolarized the membrane potential by ⬃10 mV (Fig.
phase of the voltage ramp. A larger PIC could be recorded with 7A1) and induced membrane potential oscillations within ⬃2
blockade of potassium currents by TEA and 4-aminopyridine min of application. The peak-to-peak amplitude of these oscil-
(4AP). An example is shown in Fig. 6B2, where a 10 mM TEA lations was ⬃7 mV and frequency ⬃4 Hz (Fig. 7A2). The
and 5 mM 4AP were administrated in the recording solution. A oscillations were voltage dependent with a small increase in
similar voltage ramp was applied to a type 3 EGFP⫹ neuron in frequency (1–2 Hz) when the membrane potential was depo-
lamina VII. A larger PIC (⬃100 pA) was demonstrated on the larized (Fig. 7A3) and a small decreased in the frequency (⬃2
ascending phase of the ramp while a small PIC (⬃50 pA) was Hz) when the membrane potential was hyperpolarized (A4).
also observed in the descending phase of the ramp. While PIC The amplitude of the oscillations was relatively stable when the
could be enhanced by blockade of potassium currents, poten- membrane potential was perturbed by the step currents. The
tiator of PIC could also facilitate the induction of PIC in oscillations stopped after 15 min washout (Fig. 7A, 1 and 2).
EGFP⫹ neurons, which was shown as an induction of plateau Using voltage clamp, 5-HT induced membrane current oscil-
potential with bistable firing in current clamp recording in Fig. lations were observed in another EGFP⫹ neuron in lamina VII
6C. A repetitive firing was elicited by a 2-s step current of 0.7 (Fig. 7B1). The neuron was recorded with membrane potential
nA (Fig. 6C1) in an EGFP⫹ neuron. Bath application of 12 clamped at ⫺70 mV and 2-amino-5-phosphonovaleric acid (APV,
␮M FPL64176, a L-type calcium channel potentiator, gener- 15 ␮M) and 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX, 10
ated a plateau potential with bistable firing outlasting the ␮M) applied in the bath (Fig. 7B1). Bath application of 5-HT (20
termination of the step current (Fig. 6C2). The plateau potential ␮M) induced the current oscillations with peak-to-peak amplitude
and bistable firing were blocked by 20 ␮M nifidipine (with of ⬃50 pA and frequency of ⬃6 Hz (Fig. 7B2). Of the seven
FPL64176), the L-channel antagonist (Fig. 6C3). The facilita- neurons in which 5-HT induced oscillations, two were located in
tion of plateau potential was observed in four of five EGFP⫹ lamina VII, three in lamina VIII, and two in laminar X. Five of the
neurons recorded with FPL64176 in current-clamp protocol neurons were classified as type 3, one as type 2, and one was not
from P8 –P15 cfos-EGFP mice. In a total of 67 neurons (55 tested in current clamp. The mean value of peak-to-peak ampli-
EGFP⫹ and 12 EGFP⫺) recorded with voltage-clamp protocol tude for the six cells recorded with current clamp was 8.6 mV
A
A1 A2
-68 mV -65 mV
20 mV
0 pA 0 pA
50 pA FIG. 6. Membrane currents observed in
EGFP⫹ neurons. A1: a hyperpolarization-
(Lamina X, Type 1) 200 ms
(Lamina VII, Type 3) activated inward current (Ih) was activated
by a step current of ⫺50 pA with 1-s dura-
tion injected into a type 1 cell in lamina X.
B A2: the same currents were activated with
B1 B2
same current injection in a type 3 cell in
lamina VII. Spikes elicited from postinhibi-
50 pA tory rebounds were observed in each step
100 pA after the termination of the step currents.
B1: a 5-s bivoltage ramp from ⫺70 to 0 mV
20 mV was applied to a type 3 cell in lamina VIII. A
0 mV small persistent inward current (PIC) with
1s negative current slope was demonstrated on
1s

-70 mV -70 mV
(Lamina VII, Type 3)
the rising phase of the voltage ramp. B2: the
(Lamina VIII, Type 3) similar voltage ramp was applied to a type 3
EGFP⫹ neuron in lamina VII with 10 mM
TEA and 5 mM 4-aminopyridine (4AP) ap-
plied to the recording solution. A persistent
Control
C inward current (PIC) of ⬃100 pA was dem-
C1 onstrated on the ascending phase of the ramp
while a small PIC (⬃50 pA) was also ob-
served in the descending phase of the ramp.
-65 mV C1: the repetitive firing was elicited by a 2-s
step current of 0.7 nA (bottom) in an
FPL64176 (12 M)
EGFP⫹ neuron patched in target areas.
C2: bath application of 12 ␮M FPL64176,
C2 an agonist of L-type calcium channels, gen-
erated a plateau potential with bistable fir-
-65 mV ing. C3: the bistable firing was blocked by
20 ␮M nifidipine with FPL, the L-channel
Nifidipine (20 M) + FPL antagonist.
C3
-65 mV
0.7 nA
0 pA
500 ms
(from 7 to 10 mV), and the mean frequency was 8.7 Hz (from 4 These data suggest that neuronal excitability is modestly in-
to 16.4 Hz). The 5-HT-induced membrane potential oscillations creased by 5-HT especially in the lower range of injected
appeared to be related to the neuron types (2 and 3) but not current (⬍200 pA).
laminar distribution (n ⫽ 7). The effects of 5-HT on 14 neurons (11 EGFP⫹ and 3
EGFP⫺) are summarized in Table 2. Because 5-HT-induced
5-HT modulation of cell membrane properties changes in the 11 EGFP⫹ neurons are generally agreed with
As 5-HT is critical for locomotor activity. Its effect on those in all 14 neurons (see Table 2), the data from the 14
EGFP⫹ neurons are investigated in this study. Our study neurons are used in the following presentation. Of the 14
shows that 5-HT modulated membrane properties of EGFP⫹ neurons, 5 were located in lamina VII, 4 in lamina VIII, and 5
neurons and increased neuronal excitability. A typical example in lamina X. Three were type 1, two were type 2, and nine were
is shown in Fig. 8. Repetitive firing was evoked by step type 3. The significant changes induced by 5-HT in the mem-
currents injected into a type 3 neuron in lamina VII (Fig. 8A1). brane properties included an increase in Rin (64.3 ⫾ 90 M⍀,
Bath application of 30 ␮M 5-HT generated 11 additional 18.5%), hyperpolarization of Vth (5.1 ⫾ 6 mV), increase in AP
spikes with no change in Em (Fig. 8A2). This increased excit- width (0.6 ⫾ 1 ms) and reduction in AHP depth (1.9 ⫾ 3 mV).
ability by 5-HT was accompanied by hyperpolarization of Vth Reduction in membrane potential (⫺1.3 ⫾ 7 mV), rheobase
by ⬃2.8 mV (Fig. 8A3), reduction of AHP by ⬃1.6 mV (A3), (⫺3.6 ⫾ 31 pA), AP height (⫺1.7 ⫾ 11 mV), and AHP half
and increase of Rin by ⬃7.5 M⍀. The slope of F-I relation was decay (⫺8.7 ⫾ 31 ms) were also observed in these neurons but
reduced by 38.1 Hz/nA without shift of the F-I curve (Fig. 8B). these changes were not significant.
A (Type 3, Lamina X)
A1. A2.
Control
Oscillation Washout
5 HT (20 M)
5 HT
Control
5 mV
-60 mV
1 min Washout
500 ms
A3. A4.
10 mV 10 mV
500 ms 500 ms

B (Lamina VII)
B1. Control
B2.
40 pA 20 pA
5HT (20 M)
200 ms
-70 mV
Recorded with APV (15 M) and CNQX (10 M)
1s
FIG. 7. 5-HT-induced membrane oscillations. A1: the membrane potential was maintained at about ⫺60 mV in a cell of type 3 in lamina X. About 10 mV
membrane depolarization was induced by bath application of 20 ␮M 5-HT. Membrane oscillation was observed 2 min after application of the 5-HT. The
5-HT-induced oscollation could be removed after 15 min washout. A2: the oscillation underlined by black bar in A1 was enlarged to show the details with control
and washout. The peak-to-peak amplitude of the oscillation in this cell was ⬃7 mV and frequency ⬃4 Hz. A3: a 2 s depolarizing step current with step of 50
pA was injected into the same cell. The membrane oscillation remained with a small increase (1–2 Hz) in frequency of the oscillation. A4: a 1-s hyperpolarizing
step current with step of ⫺50 pA was injected into the same cell. Again, the membrane oscillation remained but the frequency decrease by ⬃2 Hz. The last step
evoked sag current and resulted in a membrane depolarization. But the oscillation still remained. B1: 5-HT-induced membrane (current) oscillation was observed
in an EGPF neuron in lamina VII with voltage-clamp protocol. The recordings were made with membrane potential clamped at ⫺70 mV and 2-amino-5-
phosphonovaleric acid (APV, 15 ␮M) and 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX, 10 ␮M) applied to the solution. Top: control; middle: 5-HT-induced
oscillation; bottom: clamped voltage. B2: the oscillation underlined by black bar in B1 was enlarged to show the details: peak-to-peak amplitude was ⬃50 pA
and frequency ⬃6 Hz.
5-HT had a paradoxical effect on membrane potential. While determination of the Ith was increased by 50 pA at each step,
5-HT depolarized the membrane potential in 50% of the recorded therefore any changes in Ith ⬍50 pA would not be detected.
neurons (n ⫽ 14), another 50% of the neurons displayed a Data from 8 (all type 3) of the 14 cells were used to calculate
hyperpolarization of membrane potential by 5-HT. We separated the F-I relations. Five of the eight cells showed a significant
these neurons into two groups based on their membrane potential reduction in the F-I slope, accompanied by a left (n ⫽ 1) or up
response to 5-HT and summarized the effects of 5-HT on their shift (n ⫽ 4) of the F-I curves. Two of the eight cells showed
membrane properties in Table 2. The results show that although an increased in F-I slope with nonshift (n ⫽ 1) or right shift
5-HT induced a strong and opposite effect on membrane potential, (n ⫽ 1) of the F-I curves. A decrease in neuronal output was
changes in other membrane properties in these two groups of cells observed in one cell that showed a down shift of the F-I curve
were generally the same (only small, nonsignificant difference in with unchanged slope. These data demonstrate that 5-HT
AHP 1⁄2 decay). These results suggest that the variable effects of generally increased the neuronal excitability and/or enhanced
5-HT on membrane potential may not result from the changes in the output of EGFP⫹ neurons although the effects were vari-
other membrane properties measured in this study. The mecha- able.
nism underlying this paradox remains unknown. While the major
effect of 5-HT on membrane properties (mean in Table 2) were ACh modulation of cell membrane properties
observed in most of the recorded neurons, a small portion of the
neurons (⬍35%) showed no change or opposite changes in Rin, Modulation of EGFP⫹ neurons by ACh was also investi-
Vth, AP, and AHP depth and width, and 64% of the neurons gated (n ⫽ 19). A typical example is shown in Fig. 9, which
showed no change (or changes ⬍50 pA) in Ith. Note that the demonstrates recordings from a type 3 neuron in lamina VII.
measurement of Ith in this study might not reflect the real changes Bath application of ACh (20 ␮M) depolarized the membrane
in some cells because the step current injected into cells for potential by ⬃17 mV with increased background noise and
A (Type 3, lamina VII)
A1. Control A2. 5HT (30 M)
FIG. 8. 5-HT modulation of membrane

properties of EGFP⫹ neuron. A1: a repeti-
tive firing (top) was evoked in a type 3 cell
-45.2 mV -48.4 mV
in lamina VII by a 2-s step current of 50 pA
(bottom). The voltage threshold (marked by
back dots) for the first spike was ⫺45.2 mV.
A2: bath application of 30 ␮M 5-HT resulted
-66.8 mV
in a hyperpolarization of Vth in all spikes and
an elicitation of 11 more spikes with the
50 pA 10 mV same step current. The Vth was hyperpolar-
ized by 3.2 mV in the 1st spike while the
0
250 ms
resting membrane potential was almost un-
changed. A3: the 1st 5 spikes from A, 1 and
2, were averaged respectively and over-
A3. B lapped on each other (- - -, control; —,
F (Hz) 5-HT). It shows that 5-HT lowered the Vth
Control
40 Control by ⬃2.8 mV and reduce the AHP by ⬃1.6

5-HT
35 5-HT mV. B: the F-I relation was plotted for
30 steady-state firing (- - -, control; —, 5-HT).
25
The F-I slope was reduced by 38.1 Hz/nA
10 mV without shift of the F-I curve.
20
15
20 ms 10
5
0 I (pA)
0 50 100 150 200 250 300
spontaneous firing (Fig. 9A). ACh also led to a hyperpolariza- for similar observation of changes in membrane potential with
tion of Vth by ⬃4.1 mV (Fig. 9B), increase of the AHP by ⬃3.5 5-HT and ACh. Because the Ach-induced changes in the 15
mV (B), reduction of Rin by ⬃55 M⍀ (C); and lowering of the EGFP⫹ neurons are not significantly different from those in all
F-I slope by ⬃35.6 Hz/nA (D). Note that the Ih was also 19 neurons (see Table 3), the data from the 19 neurons are used
blocked by ACh in this neuron (Fig. 9C). The change in the F-I in the following presentation. Of 19 recorded cells, 4 were
relation suggests that the excitability of the cell was increased located in lamina VII (1 type 1 and 3 type 3), 4 in lamina VIII
by ACh in the lower range of injected current ⱕ250 pA but (1 type 1, 1 type 2, and 2 type 3), and 11 in lamina X (1 type
reduced in the higher range over 250 pA. This “current- 1, 5 type 2, and 5 type 3). Although ⬎50% of the cells were
dependent” modulation of neuronal excitability might result distributed in lamina X and the majority of the cells were type
from a nonlinear balance of excitatory (slow depolarization of 3, the ACh-induced changes in membrane properties were not
Em and lowering of Vth) and inhibitory (increase of AHP and significantly related to either laminar distribution or cell type.
reduction of Rin and Ih) forces driven by ACh. The significant changes induced by ACh include a depolariza-
The effects of ACh on neuronal membrane properties from tion of Em (3.7 ⫾ 9 mV), reduction of Rin (⫺56.4 ⫾ 78 M⍀,
19 neurons (15 EGFP⫹ and 4 EGFP⫺) are summarized in 13.2%), hyperpolarization of Vth (⫺3.9 ⫾ 4 mV), reduction of AP
Table 3. The table is organized in the same format as Table 2 amplitude (⫺5.5 ⫾ 12 mV) with an increase in AP width (0.3 ⫾
TABLE 2. 5-HT-induced changes in cell membrane properties
Changes in Membrane Properties
Control Mean
Total EGFP⫹ Total EGFP⫹ 1Em Total 2Em Total
n 14 11 14 11 7 7
Em, mV ⫺72.3 ⫾ 8 ⫺72.9 ⫾ 8 ⫺1.3 ⫾ 7 ⫺0.8 ⫾ 5 4.5 ⫾ 3* ⫺7.1 ⫾ 3*
Ith, pA 107.1 ⫾ 61 118.2 ⫾ 64 ⫺3.6 ⫾ 31 ⫺4.5 ⫾ 35 ⫺7.1 ⫾ 34 0.0 ⫾ 29
Rin, M⍀ 347.8 ⫾ 111 342.5 ⫾ 123 64.3 ⫾ 90* 71.5 ⫾ 99* 70.6 ⫾ 54* 58.0 ⫾ 121
Vth, mV ⫺39.6 ⫾ 10 ⫺39.7 ⫾ 11 ⫺5.1 ⫾ 6* ⫺3.4 ⫾ 4† ⫺2.4 ⫾ 4† ⫺8.3 ⫾ 6†
AP height, mV 43.5 ⫾ 13 39.2 ⫾ 11 ⫺1.7 ⫾ 11 ⫺3.4 ⫾ 11 ⫺2.5 ⫾ 13 ⫺0.7 ⫾ 9
AP width, ms 3.8 ⫾ 1 3.9 ⫾ 1 0.6 ⫾ 1† 0.6 ⫾ 1† 0.4 ⫾ 1 0.7 ⫾ 1†
AHP depth, mV 16.4 ⫾ 8 15.8 ⫾ 8 ⫺1.9 ⫾ 3† ⫺1.7 ⫾ 3† ⫺1.5 ⫾ 4 ⫺2.4 ⫾ 2†
AHP 1/2 decay, ms 52.1 ⫾ 43 46.3 ⫾ 27 ⫺8.7 ⫾ 31 ⫺5.6 ⫾ 21 ⫺17.9 ⫾ 39 2 ⫾ 13
Slope SS, Hz/nA 88.3 ⫾ 37 88.1 ⫾ 39 ⫺11.1 ⫾ 37 ⫺20.6 ⫾ 27 ⫺19.2 ⫾ 29 ⫺3.1 ⫾ 47
1, depolarization (ⱖ2 mV in average); 2, hyperpolarization (ⱕ⫺2 mV in average); †, significant difference with P ⬍ 0.05 and *, P ⬍ 0.005 performed by
paired two-sample t-test.

A (Type 3, lamina VII)

20 mV
ACh (20 M) FIG. 9. ACh modulation of membrane properties of
EGFP⫹ neuron. A: recordings were made from a type 3 cell
in lamina VII. A: 20 ␮M ACh was applied to the bath
-80 mV
solution (2), and the membrane potential was depolarized
10 s
by ⬃17 mV after 1-min application of the drug. Spontane-
ous firing occurs occasionally during the depolarization of
the membrane potential. B1: the hyperpolarization of Vth
B and increase of afterhyperpolarization (AHP) were shown
B2. in the repetitive firings evoked by a 2-s ramp current
B1.
starting from 0 pA. Spike trains from control (- - -) and
20 mV
ACh (—) were overlapped with unchanged baseline for
-40.1 mV Control 10 mV resting membrane potentials. Voltage thresholds were
-44.2 mV ACh marked by dots on the spikes (black for control and open for
10 ms ACh). B2: the 1st 5 spikes from both control and ACh
conditions were averaged, respectively, and overlapped on
250 pA
each other (- - -, control; —, Ach) to show the changes in
0 200 ms Vth, AHP and action potential (AP) in this neuron. C: a
hyperpolarizing current (1 s, ⫺100 pA) was injected into
the same cell before and after the ACh application. Mem-
brane potential deflections from the control (- - -) and ACh
C D (—) conditions were overlapped and aligned to the resting
F (Hz)

35 membrane potentials. Spikes were elicited from postinhibi-
Control Control tory rebound in both conditions. ACh hyperpolarized the
30
ACh
10 mV
25
ACh
Vth by ⬃4.1 mV, reduced input resistance by ⬃55 M⍀ and
increased the AHP by ⬃3.5 mV in this neuron. D: the F-I
20
curves were plotted for steady-state spikes. ACh reduced
15 the F-I slope by ⬃35.6 Hz/nA. The excitability of the cell
10 was increased in the lower range of injected current (ⱕ250
5 pA) but slightly reduced in the higher range over 250 pA.
200 ms
-100 pA 0 I (pA)
0 50 100 150 200 250 300 350
0.7 ms), and an increase of AHP depth (1.1 ⫾ 3 mV) with an and increase of F-I slope could be directly induced by the
increase in AHP half-decay time (3.8 ⫾ 15 ms). Similar to the depolarization of Em in the group of depolarizing neurons,
case of 5-HT, ACh also induced paradoxical changes in mem- whereas the hyperpolarization of Em could lead to the opposite
brane potential. Of the 19 cells, 10 cells (52%) showed depolar- effects on rheobase and F-I slope in the group of hyperpolarizing
ization of Em (mean ⫽ 10.4 ⫾ 7 mV), 6 cells (31%) displayed neurons. Similar to the experiments of 5-HT, a small portion
hyperpolarization of Em (mean ⫽ ⫺5.7 ⫾ 5 mV), and 3 cells of the neurons (⬍35%) displayed no change or opposite
(15%) did not respond to ACh in Em. Both changes (depolariza- changes in Rin, Vth, AP, and AHP properties, and a large
tion and hyperpolarization) were statistically significant. Except number of the cells (68%) showed no change (or changes
for the differences in rheobase and slope of the F-I relation, all ⬍50 pA) in rheobase.
other changes in membrane properties in these two groups of The F-I relations were calculated in 11 of the 19 neurons (8
neurons were very similar (see Table 3), suggesting that the type 3 and 3 type 2). Of the 11 neurons, 64% of the cells (7/11)
mechanism underlying the changes in membrane potential by showed a significant reduction of the F-I slope with left (n ⫽
ACh may not result from the changes in other properties shown in 3), up (n ⫽ 3), or down shift (n ⫽ 1) of the F-I curves. 36%
the Table 3. Instead the changes in membrane potential might (4/11) of the cells showed an increase in the F-I slope with
cause the changes in Ith and F-I slope. The reduction of rheobase nonshift (n ⫽ 1), down (n ⫽ 2), or up shift (n ⫽ 1) of the F-I
TABLE 3. ACh-induced changes in cell membrane properties
Changes in Membrane Properties
Control Mean
Total EGFP⫹ Total EGFP⫹ 1Em Total 2Em Total
n 19 15 19 15 10 6
Em, mV ⫺68.7 ⫾ 7 ⫺69.3 ⫾ 7 3.7 ⫾ 9† 3.1 ⫾ 9† 10.4 ⫾ 7* ⫺5.7 ⫾ 5†
Ith, pA 73.7 ⫾ 31 66.7 ⫾ 24 2.6 ⫾ 48 10.0 ⫾ 48 ⫺10.0 ⫾ 39 25 ⫾ 69
Rin, M⍀ 426.7 ⫾ 241 446.7 ⫾ 242 ⫺56.4 ⫾ 78* ⫺69.6 ⫾ 78† ⫺60.9 ⫾ 92† ⫺37.2 ⫾ 68
Vth, mV ⫺40.7 ⫾ 10 ⫺41.8 ⫾ 8 ⫺3.9 ⫾ 4* ⫺3.3 ⫾ 3* ⫺3.4 ⫾ 4† ⫺5.6 ⫾ 3*
AP height, mV 50.4 ⫾ 13 50.3 ⫾ 12 ⫺5.5 ⫾ 12† ⫺5.6 ⫾ 12† ⫺6.6 ⫾ 16 ⫺2.8 ⫾ 10
AP width, ms 3.3 ⫾ 1 3.1 ⫾ 1 0.3 ⫾ 1† 0.3 ⫾ 0.7† 0.4 ⫾ 1 0.2 ⫾ 0.3
AHP depth, mV 14.1 ⫾ 4 14.0 ⫾ 5 1.1 ⫾ 3† 0.8 ⫾ 3† 1.4 ⫾ 4 0.5 ⫾ 2
AHP 1/2 decay, ms 51.6 ⫾ 30 50.7 ⫾ 30 3.8 ⫾ 15 3.0 ⫾ 15 0.4 ⫾ 17 5.9 ⫾ 15
Slope SS, Hz/nA 94.8 ⫾ 30 99.1 ⫾ 29 4.8 ⫾ 52 12.2 ⫾ 52 12.5 ⫾ 59 ⫺2.4 ⫾ 5
1, depolarization (ⱖ2 mV in average); 2, hyperpolarization (ⱕ⫺2 mV in average); †, significant difference with P ⬍ 0.05 and *, P ⬍ 0.005 performed by
paired two-sample t-test.

curve. These changes in F-I relation demonstrate the ACh ACh did not reduce the ability of 5-HT to increase Rin,
modulation of neuronal excitability and output through a bal- consistent with the notion that these two agents may alter Rin
ance of excitatory and inhibitory forces driven by ACh in the via different mechanisms. ACh hyperpolarized the Vth by
EGFP⫹ neurons. ⫺3.4 ⫾ 4 mV, whereas 5-HT further lowered it by an addi-
tional ⫺4.3 ⫾ 5 mV. Thus prior application of ACh did not
Combined effects of 5-HT and ACh on EGFP⫹ neurons diminish the ability of 5-HT to hyperpolarize voltage thresh-
old, suggesting that the two agents may act by different
The combined effects of 5-HT and ACh on cell membrane mechanisms to achieve the same effect on Vth. Similarly, ACh
properties were investigated in this study to examine the increased AHP depth (0.1 ⫾ 2 mV) and width (1.1 ⫾ 20 ms),
relative mechanisms mediating the modulation of membrane whereas 5-HT reduced this (⫺1.9 ⫾ 3 mV; ⫺6.7 ⫾ 10 ms). In
properties by the two agents. Figure 10 shows modulation of another five cells, the inverse sequence of drug application was
neuronal properties by these agents with recordings made from used. 5-HT increased the Rin (24.0 ⫾ 83 M⍀), hyperpolarized
a type 3 neuron in lamina VIII. In control, the Rin was ⬃540 Vth (⫺7.0 ⫾ 5 mV), and reduced AHP (⫺2.0 ⫾ 1 mV),
M⍀ (Fig. 10A1) and Vth was ⫺35.0 mV (A2). Bath application whereas ACh applied subsequent to 5-HT reduced Rin
of 20 ␮M ACh reduced the Rin by ⬃190 M⍀ (Fig. 10B1) and (⫺52.4 ⫾ 41 M⍀), further hyperpolarized the Vth (⫺5.6 ⫾ 8
hyperpolarized the Vth by 8.6 mV (B2). Subsequent application mV), and increased AHP (2.2 ⫾ 2 mV).
of 20 ␮M 5-HT increased the Rin by ⬃180 M⍀ from 350 M⍀ In general, the effects of 5-HT after ACh or the effects of
measured in ACh condition (Fig. 10C1) and further hyperpo- ACh after 5-HT were consistent with those of 5-HT and
larized the Vth by 9.1 mV from ⫺43.6 mV measured in ACh ACh alone on the EGFP⫹ neurons as listed in Tables 2 and

condition (C2). 5-HT also induced excitatory postsynaptic 3. Thus neither agent alters the modulatory properties of the
potentials (EPSPs) in this cell (Fig. 10C1). This figure also other on EGFP⫹ neurons, consistent with the suggestion
provides evidence for a reduction of Ih by ACh (Fig. 10B1). that their actions on membrane properties of these cells are
Interestingly, this reduction is reversed by subsequent ap- exerted by separate mechanisms. The additive effects of
plication of 5-HT (Fig. 10C1). A similar reduction (or 5-HT and ACh on Vth would increase neuronal excitability,
blockade) of Ih by ACh is also shown in Fig. 9C. These whereas the subtractive effects of 5-HT and ACh on Rin and
results demonstrate that the effects of 5-HT and ACh on AHP would either increase or decrease the cell excitability
EGFP⫹ neurons can be additive (e.g., Vth) or subtractive depending on the balance of the forces driven by the two
(e.g., Rin and Ih), depending on the specific membrane agents.
properties they modulate.
Eleven cells were investigated for combined modulation of Changes in membrane potential with modulation of intrinsic
cell membrane properties by 5-HT and ACh. Six cells were membrane properties
recorded with application of ACh followed by 5-HT. ACh
reduced the Rin by ⫺63.5 ⫾ 134 M⍀, and subsequent appli- Both 5-HT and ACh induced variable effects on mem-
cation of 5-HT reversed this reduction of Rin and led to an brane potential. Because Em and Rin were both shown to be
increase in Rin by 84.3 ⫾ 147 M⍀. Thus prior application of modulated by 5-HT (Table 2) and ACh (Table 3), we would
(Type 3, lamina VIII)
A B C
A1 B1 C1
Control Ach (20 M) 5-HT (20 M)
-65.4 mV
Rin=350 M
Rin=540 M Rin=530 M
20 mV
0 pA
500 ms
-100 pA
A2 B2 C2
-35.0 mV
-43.6 mV -52.7 mV
-68.2 mV
20 mV
0 pA 200 pA
100 ms
FIG. 10. Combined effects of ACh and 5-HT on EGFP⫹ neurons. Recordings were made from a type 3 cell in lamina VIII in control (A) and conditions of
ACh (B) and 5-HT (C). The current injections in A–C were the same as those shown in A. A: control recordings. A1: hyperpolarizing step current (1 s with 50
pA for each step, bottom traces) was injected into the cell. Voltage deflection (top traces) produced by the ⫺50-pA step current was used to calculate Rin (540
M⍀). A2: repetitive firing (top trace) was evoked in the same cell by a 2-s ramp current (bottom trace). The Vth was measured from the 1st spike (⫺35.0 mV).
B: recordings with bath application of 20 ␮M ACh. B1: the Rin (358 M⍀) was reduced by 182 M⍀ and Ih was diminished. B2: in the same condition of ACh
the Vth (⫺43.6 mV) of the 1st spike was hyperpolarized by 8.6 mV. C: recordings with bath application of 5-TH (20 ␮M) subsequently. C1: 5-HT increased
the Rin (530 M⍀) by 172 M⍀ from that measured in the condition with ACh. C2: Vth (⫺52.7 mV) was further hyperpolarized by 9.1 mV by 5-HT.

expect that the changes induced by 5-HT and ACh in Em tern. As shown in other systems, such as the stomatogastric
could result from the changes in Rin. Figure 11 shows the ganglion (Marder and Bucher 2001), it is necessary to under-
correlations between Em and Rin and the changes induced by stand neuronal connections, their intrinsic properties, and the
5-HT (n ⫽ 14) and ACh (n ⫽ 19). No significant correlation modulation of these properties to understand rhythm genera-
was found between Em and Rin (Fig. 11, A1 and B1) before tion. In the adult cat, interneurons can be identified by their
or after administration of 5-HT (correlation coefficient R ⫽ location and synaptology (Jankowska 2008). However, the
0.24 for control, 0.40 for 5-HT) or ACh (R ⫽ 0.11 for intrinsic and modulatable properties of these neurons have not
control, 0.43 for ACh). These results suggested that the been studied. In the mouse, interneurons can be identified
resting membrane potential was not regulated simply by the through labeling with GFP driven by, for example, transcrip-
passive membrane properties such as the size of EGFP⫹ tion factors expressed during development (e.g., Wilson et al.
neurons. Also, the ⌬Em was not correlated to the ⌬Rin in
2005; Zhang et al. 2008), or through retrograde labeling with
either 5-HT (R ⫽ 0.05, Fig. 11A2) or ACh (R ⫽ 0.15, Fig.
dyes (Carlin et al. 2006; Eide et al. 1999; Stokke et al. 2002).
11B2) condition. Furthermore, the changes in Em and Rin
With these techniques, the intrinsic membrane properties of the
were neither correlated to the laminar distributions of the
neurons nor to the types of the neurons (not shown). These identified neurons can be studied directly in either in vitro or in
results suggest that modulation of some unknown active vivo preparations. The present study demonstrates a method of
(rather than passive) membrane properties by 5-HT and ACh studying spinal interneurons that are functionally activated by
result in the observed changes in membrane potentials in locomotor activities and visualized by EGFP expression driven
EGFP⫹ neurons. by the promoter for the immediate early gene, c-fos. The
membrane properties of the EGFP⫹ neurons and their modu-

latory properties by neurotransmitters 5-HT and ACh are
DISCUSSION
studied in detail in this study. A conceptually similar circadian
In this study, we demonstrate a new method by which clock gene promoter-driven d2EGFP transgenic reporter
neurons active in locomotion can be identified and their intrin- mouse line has been used to target and study functionally
sic properties studied in the mouse spinal cord. Identification of activated biological clock neurons and circadian modulation of
spinal interneurons involved in locomotion is a necessary step locomotor behavior (Ciarleglio et al. 2009; Kuhlman et al.
to understand how the spinal cord produces rhythm and pat- 2003; Quintero et al. 2003).
A Correlation between Em and Rin and changes induced by 5-HT
A1. A2.
Rin (M ) Em (mV)
0 200 400 600 800 1000 15
-50
-55 10
Control
-60 5HT
5
-65
Em (mV)
-70 0 2
R = 0.002
-75 R2= 0.060
-5
-80 FIG. 11. Correlation analysis between membrane poten-
-85 tial and input resistant. A: correlations between Em and Rin
-10
in both control (E - -) and 5-HT (F –) conditions are estab-
-90
R2= 0.161 -15 lished from 14 cells in A1. Changes induced by 5-HT in Em
-100 and Rin in the same group of neurons are plotted in A2.
-100 0 100 200 300 400
B: similar to A, the Em is correlated with Rin in both control
Rin (M ) (E - -) and ACh (F –) conditions from the recordings of 19
cells in B1. Changes induced by ACh in Em and Rin are
B Correlation between Em and Rin and changes induced by ACh shown in B2. None of the preceding relations is significantly
correlated, suggesting that rather than passive membrane
B1. B2. properties, the 5-HT and ACh induced changes in Em and
Em (mV) Rin may mainly result from modulation of active membrane
Rin (M ) properties of the EGFP⫹ neurons.
0 200 400 600 800 1000 30
-50
-55 20
-60 2
R = 0.012
Em (mV)
-65 10
-70 2
R = 0.024
-75 0
2
R = 0.188
-80
-85
-10
-90 Control ACh 0
-300 -200 -100 100
Rin (M )

Expression of c-fos-EGFP in spinal interneurons fashion by the neurotransmitters ACh and 5-HT. These simi-
larities include the depolarization of membrane potential and
In addition to locomotor task, c-fos expression can be also reduction of input resistance by ACh, and the depolarization of
induced by many other factors (Coggeshall 2005) such as cell membrane potential, reduction of AHP, hyperpolarization of
death and surgical procedures in spinal neurons. To reduce
Vth, reduction of rheobase, and increase of input resistance and
these effects on our study, we have set several criteria to induce action potential width by 5-HT. In addition, the target areas
c-fos expression and to choose the EGFP⫹ neurons in this (laminar VII, VIII, and X) for patching the EGFP⫹ neurons are
study: 1) the locomotor task (swimming) was induced in overlapped with the areas where the ascending commissural
animals for 60 –90 min. This long-duration, persistent, and interneurons locate (Carlin et al. 2006; Eide et al. 1999). It is
nonstopping locomotor activity would allow spinal neurons therefore possible that a proportion of the EGFP⫹ neurons
driven by the locomotion to have enough time and intensity to studied here are ascending commissural interneurons.
express c-fos (Coggeshall 2005). In fact, our results confirmed
the validity of this protocol for induction of the c-fos-EGFP
Membrane currents in EGFP⫹ neurons
expression in spinal interneurons (Fig. 3). 2) Apoptosis is
known to be associated with c-fos expression (Smeyne et al. Two important ionic currents were found in EGFP⫹ neurons
1993). In spinal interneurons, the death of cells due to apopto- in the present study: hyperpolarization-activated inward cur-
sis reached the peak at P0 and decreased rapidly by ⬃80% after rents and persistent inward currents. Ih has been shown to be
P4 (Lowrie and Lawson 2000). For this reason, the animals we involved in the production of bursting during various forms of
chose for patch-clamp experiments were all at (16%, 7/44) or rhythmic activity (Bertrand and Cazalets 1998; Butt et al.
older (84%, 37/44) than P4. This selection of animals could 2002; Kiehn et al. 2000; Thoby-Brisson et al. 2000) while PICs

reduce the effect of apoptosis on cfos-EGFP expression. have been also shown to play an important role in rhythm
3) Although surgical procedures such as dissection and slicing generation in respiration (Del Negro et al. 2002, 2005) and
process could induce cfos expression, neurons expressing c- locomotion (Tazerart et al. 2008; Zhong et al. 2007). The
fos-EGFP by this way are usually unhealthy. This can be expression of Ih and PICs in EGFP⫹ neurons supports the
recognized by the shrinking cell body, visualized nuclear, importance of these two currents in the generation of locomo-
rough surface, and ultra brightness of the EGFP expression. tion. However, further studies are required to characterize Ih
These cells were excluded for patch-clamp recording. In this and PICs in EGFP⫹ neurons.
study, only healthy neurons that were usually shown as me-
dium (or sometimes weak) brightness of EGFP expression in 5-HT-induced membrane oscillations
target areas (lamina VII, VIII, and X) were chosen for patch-
clamp study. Although the number of EGFP⫹ neurons induced 5-HT-induced membrane oscillation was observed in
by nonlocomotor activity could be largely reduced by these EGFP⫹ neurons. Similar observations have been reported in
criteria, we could not absolutely rule out the c-fos-EGFP recent studies in ascending commissural interneurons in neo-
expression triggered by some unwanted factors. The EGFP⫹ natal mice (Carlin et al. 2006; Zhong et al. 2006a), where the
neurons that can be always seen in dorsal horn in both control 5-HT-induced membrane oscillations were shown to be either
and locomotor slices are such an example. As shown in Fig. 3, voltage dependent (Carlin et al. 2006) or independent (Zhong
however, the pronounced increase in EGFP expression in et al. 2006a) and could be blocked by TTX or the gap junction
ventral horn after swimming suggested that the locomotor blocker carbenoxolone (Zhong et al. 2006a), suggesting that
activity indeed induced EGFP expression in spinal interneu- multiple mechanisms could be responsible for the oscillations.
rons. These neurons are the target of the present study. In this study, we demonstrated that in some cells, the frequency
of the oscillations was voltage dependent (Fig. 7A) and could
Differences among the firing patterns, lamina distributions, be persisted with APV and CNQX in the recording solution
and membrane properties (B). These results indicate that intrinsic membrane properties
play a role in generating the 5-HT-mediated membrane poten-
A significant difference in Rin and Cm was shown to be tial oscillations in some EGFP⫹ neurons, which may therefore
correlated with neuronal laminar distribution but not type. This have a role in rhythm generation. Membrane oscillation re-
suggests differences in size and/or morphology of the activated quires a coupling of inward and outward currents with precise
neurons in the target areas. The active properties (Vth, AP, occurrence of the currents in time. As suggested by many
AHP, and F-I properties, etc), however, were not significantly previous studies (e.g., Marder and Bucher 2001; Wilson et al.
related to the laminar distribution. Therefore the electrophys- 2005), Ih, T-type calcium currents, and PICs (calcium and/or
iological or functional differences in these neurons with respect sodium components) are all candidates for induction of mem-
to their role in locomotion is not clear. brane oscillations in locomotor neurons. Enhancement of Ih
and PICs by 5-HT have been reported in our recent studies (Dai
Overlapping of the EGFP⫹ neurons with some identified and Jordan 2006; Dai et al. 2005a). Therefore the action of
locomotor neurons 5-HT on Ih and PICs could facilitate the membrane oscillation
in EGFP⫹ neurons observed in this study. Further study is
The functional role of the EGFP⫹ neurons in locomotion is required to investigate this issue.
unknown. It is not possible to classify the EGFP⫹ neurons into
any known type of locomotor interneuron given differences in Aminergic effects on EGFP⫹ neurons
the types of studies. However, the EGFP⫹ neurons and as-
cending commissural interneurons previously studied (Carlin In this study, we demonstrate the modulation of membrane
et al. 2006; Zhong et al. 2006a,b) are modulated in a similar properties by 5-HT and ACh in EGFP⫹ neurons. Although
both agents increase the neuronal excitability, their effects on Butt SJ, Lundfald L, Kiehn O. EphA4 defines a class of excitatory locomo-
membrane properties are not exactly the same. Both agents tor-related interneurons. Proc Natl Acad Sci USA 102: 14098 –14103, 2005.
Carlin KP, Dai Y, Jordan LM. Cholinergic and serotonergic excitation of
induced paradoxical effects on membrane potential and hyper- ascending commissural neurons in the thoraco-lumbar spinal cord of the
polarized Vth. On the other side, however, 5-HT increased Rin neonatal mouse. J Neurophysiol 95: 1278 –1284, 2006.
and reduced AHP, whereas ACh reduced Rin and increased Carlin KP, Jones KE, Jiang Z, Jordan LM., Brownstone RM. Dendritic
AHP. The variable effects of 5-HT and ACh on EGFP⫹ L-type calcium currents in mouse spinal motoneurons: implications for
neurons could result in different mechanisms to regulate the instability. Eur J Neurosci 12: 1635–1646, 2000.
neuronal excitability and output. 5-HT-induced changes in F-I Cazalets JR, Borde M, Clarac F. Localization and organization of the central
pattern generator for hindlimb locomotion in newborn rat. J Neurosci 15:
slope (n ⫽ 7) tended to be correlated to changes in Rin
4943– 4951, 1995.
(absolute value of correlation coefficient R ⫽ 0.6), AHP depth Cazalets J-R, Borde M, Clarac F. The synaptic drive from the spinal
(R ⫽ 0.6), and Vth (R ⫽ 0.8), whereas ACh-induced changes in locomotor network to motoneurons in the newborn rat. J Neurosci 16:
F-I slope (n ⫽ 11) were coupled relatively strongly to changes 298 –306, 1996.
in Rin (R ⫽ 0.7) and AP height and width (R ⫽ 0.5) but weakly Cazalets JR, Sqalli-Houssaini Y, Clarac F. Activation of the central pattern
to AHP duration (R ⫽ 0.3) and Vth (R ⫽ 0.2). These differences generators for locomotion by serotonin and excitatory amino acids in
in correlation reflected different levels of balance in modula- neonatal rat. J Physiol 455: 187–204, 1992.
Ciarleglio CM, Gamble KL, Axley JC, Strauss BR, Cohen JY, Colwell CS,
tory forces driven by different mechanisms. McMahon DG. Population encoding by circadian clock neurons organizes
circadian behavior. J Neurosci 29: 1670 –1676, 2009.
Conclusion Cina C, Hochman S. Diffuse distribution of sulforhodamine-labeled neurons
during serotonin-evoked locomotion in the neonatal rat thoracolumbar

Using cfos-EGFP transgenic mice, we demonstrate the abil- spinal cord. J Comp Neurol 423: 590 – 602, 2000.
ity to study interneurons activated by a locomotor task. The Coggeshall RE. Fos, nociception and the dorsal horn. Prog Neurobiol 77:
299 –352, 2005.
capacity to select neurons active during locomotion adds a new
Cowley KC, Schmidt BJ. A comparison of motor patterns induced by
method in which interneurons can be studied and may be N-methyl-aspartate, acetylcholine and serotonin in the in vitro neonatal rat
particularly powerful when used in combination with other spinal cord. Neurosci Lett 171: 147–150, 1994.
techniques such as retrograde labeling technique for study of Cowley KC, Schmidt BJ. Regional distribution of the locomotor pattern-
commissural interneurons specifically activated by locomotion. generating network in the neonatal rat spinal cord. J Neurophysiol 77:
Characterization of EGFP⫹ neurons located in lamina VII, 247–259, 1997.
VIII, and X is important to provide insight into the cellular Crone SA, Quinlan KA, Zagoraiou L, Droho S, Restrepo CE, Lundfald L,
Endo T, Setlak J, Jessell TM, Kiehn O, Sharma K. Genetic ablation of
basis for generation of locomotion. The membrane properties V2a ipsilateral interneurons disrupts left-right locomotor coordination in
of these neurons and the manner in which they are modulated mammalian spinal cord. Neuron 60: 70 – 83, 2008.
by 5-HT and ACh suggest that the EGFP⫹ neurons could play Dai Y, Jordan LM. Characterization of persistent inward currents (PICs) in
a role in generating or mediating locomotion. locomotor activity-related neurons of Cfos-EGFP mice. Soc Neurosci Abstr
130.6, 2006.
ACKNOWLEDGMENTS Dai Y, Jordan LM, Brownstone RM. Characterization of electrophysiolog-
ical and pharmacological properties of locomotor activity related neurons in
We thank J. McVagh, C. Gibbs, G. Detillieux, M. Ellis, J. Rutherford, and cfos-egfp mice. Soc Neurosci Abstr 882.14, 2004.
M. Setterbom for technical support. Dai Y, Jordan LM, Brownstone RM. Characterization of hyperpolarization-
activated inward current in locomotor activity related neurons in Cfos-EGFP
GRANTS mice. Soc Neurosci Abstr 377.3, 2005a.
This work is supported by National Institutes of Health Grant 391313520 Dai X, Noga BR, Douglas JR, Jordan LM. Localization of spinal neurons
and the Canadian Institute of Health Research to L. M. Jordan. activated during locomotion using the c-fos immunohistochemical method.
J Neurophysiol 93: 3442–3452, 2005b.
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