J Neurophysiol 105: 235–248, 2011.
First published November 3, 2010; doi:10.1152/jn.00493.2010.
GAD67-GFP⫹ Neurons in the Nucleus of Roller: A Possible Source
of Inhibitory Input to Hypoglossal Motoneurons. I. Morphology
and Firing Properties
J.F.M. van Brederode,1 Y. Yanagawa,2,3 and A. J. Berger1
1
Department of Physiology and Biophysics, University of Washington, Seattle, Washington; 2Department of Genetic and Behavioral
Neuroscience, Gunma University Graduate School of Medicine, Maebashi; and 3Japan Science and Technology Agency, Collaboration
of Regional Entities on Science and Technology, Tokyo, Japan
Submitted 1 June 2010; accepted in final form 3 November 2010
INTRODUCTION
Hypoglossal motoneurons (HMs) are a diverse group of
brain stem neurons that innervate the tongue muscles. The
tongue is involved in various basic tasks such as sucking,
mastication, swallowing, and vocalization. The tongue is also
active in respiration and controls upper airway patency. HMs
are thought to play a role in the pathogenesis of obstructive
sleep apnea (Horner 2007). Since HMs are involved in a
variety of oropharyngeal behaviors, it is not surprising that
they are controlled by a host of brain stem neural networks,
including inhibitory networks. The role of synaptic inhibition
Address for reprint requests and other correspondence: H. van Brederode,
UW Physiology and Biophysics, 1705 NE Pacific St., HSB G424, Box 357290,
Seattle, WA 98195-7290 (E-mail: hansvb@washington.edu).
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in generating HM output is not well understood, however.
Functionally, synaptic inhibition has been shown to contribute
to inspiratory but not expiratory HM membrane potential
trajectories (Saywell and Feldman 2004; Withington-Wray et
al. 1988; Woch and Kubin 1995). Exogenously applied ␥-aminobutyric acid (GABA) or glycine depresses spike firing of
HMs stimulated with intracellular current pulses (Marchetti et
al. 2002). There is also a significant tonic component to the
synaptic inhibition received by HMs (Paton and Richter 1995)
and blockade of this tonic inhibition results in an increase in
input resistance and membrane time constant in HMs (NunezAbades et al. 2000).
Both glycinergic and GABAergic synaptic terminals are
found on the somata and dendrites of HMs (Aldes et al. 1988).
The immunostaining for glutamic acid decarboxylase (GAD) is
most dense in the ventromedial part of the nucleus (Aldes et al.
1988), a region that contains predominantly motoneurons that
innervate the genioglossus muscle of the tongue (Krammer et
al. 1979). By combining patch-clamp recordings and immunocytochemistry it was shown that HMs from the ventrolateral
part of the XII nucleus have spontaneous miniature inhibitory
synaptic currents with a large GABAA receptor component and
HMs in this part of the nucleus show more dense labeling for
GABAA receptors than HMs in other parts of the nucleus
(O’Brien and Berger 2001). These findings suggest that GABA
innervation of the XII nucleus is not uniform, but is strongest
in the ventral part of the nucleus. The source of these inhibitory
synaptic terminals is not known, but they are believed to
originate from the reticular formation adjacent to the XII
nucleus (Donato and Nistri 2000; Umemiya and Berger 1995)
or from interneurons located within the XII nucleus itself
(Takasu and Hashimoto 1988). Immunocytochemistry for
GABA and its synthesizing enzyme GAD has revealed that a
population of inhibitory neurons is clustered just ventral to the
XII nucleus (Aldes et al. 1988) in and around the Nucleus of
Roller (NR). Combined retrograde labeling and GAD immunocytochemstry indicates that inhibitory premotor neurons that
innervate HMs are located in this part of the reticular formation
(Li et al. 1997). Local stimulation of the NR elicits glycinergic
inhibitory postsynaptic currents (IPSCs) in HMs (Hulsmann et
al. 2000), but it is not known whether GABAergic IPSCs can
be evoked by stimulation of the area. It has been suggested that
interneurons in the NR inhibit HMs as part of the trigeminohypoglossal reflex (Sumino and Nakamura 1974). Most HMs
receive synaptic inhibition during inspiration and this inhibi-
0022-3077/11 Copyright © 2011 The American Physiological Society
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van Brederode JFM, Yanagawa Y, Berger AJ. GAD67-GFP⫹
neurons in the Nucleus of Roller: a possible source of inhibitory input
to hypoglossal motoneurons. I. Morphology and firing properties. J
Neurophysiol 105: 235–248, 2011. First published November 3, 2010;
doi:10.1152/jn.00493.2010. In this study we examined the electrophysiological and morphological properties of inhibitory neurons
located just ventrolateral to the hypoglossal motor (XII) nucleus in the
Nucleus of Roller (NR). In vitro experiments were performed on
medullary slices derived from postnatal day 5 (P5) to P15 GAD67GFP knock-in mouse pups. ON cell recordings from GFP⫹ cells in NR
in rhythmic slices revealed that these neurons are spontaneously
active, although their spiking activity does not exhibit inspiratory
phase modulation. Morphologically, GFP⫹ cells were bi- or multipolar cells with small- to medium-sized cell bodies and small dendritic trees that were often oriented parallel to the border of the XII
nucleus. GFP⫹ cells were classified as either tonic or phasic based on
their firing responses to depolarizing step current stimulation in whole
cell current clamp. Tonic GFP⫹ cells fired a regular train of action
potentials (APs) throughout the duration of the pulse and often
showed rebound spikes after a hyperpolarizing step. In contrast,
phasic GFP⫹ neurons did not fire throughout the depolarizing current
step but instead fired fewer than four APs at the onset of the pulse or
fired multiple APs, but only after a marked delay. Phasic cells had a
significantly smaller input resistance and shorter membrane time
constant than tonic GFP⫹ cells. In addition, phasic GFP⫹ cells
differed from tonic cells in the shape and time course of their spike
afterpotentials, the minimum firing frequency at threshold current
amplitude, and the slope of their current–frequency relationship.
These results suggest that GABAergic neurons in the NR are morphologically and electrophysiologically heterogeneous cells that could
provide tonic inhibitory synaptic input to HMs.
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J.F.M. VAN BREDERODE, Y. YANAGAWA, AND A. J. BERGER
METHODS
Experimental procedures
In vitro slice experiments were performed on medullary slices
derived from heterozygous neonatal (P5 to P15) GAD67-GFP
knock-in mouse pups (Tamamaki et al. 2003). Since both the GFP and
the GAD67 genes in the knock-in mice are identically controlled, the
parallel GFP protein expression accurately reflects the expression of
GAD67 in GABA neurons (Acuna-Goycolea et al. 2005; Brown et al.
2008; Ono et al. 2005; Tamamaki et al. 2003). Newborn pups were
phenotyped on postnatal days P1–P3 by examining the head of the
mouse using light from a “miner’s lamp” apparatus (Model GFsP-5;
BLS, Budapest). With this device the brains and olfactory bulbs (as
visualized through the thin skull of the newborn) of GAD67-GFP
knock-in mice glowed green (Brown et al. 2008). Those whose brains
and olfactory bulbs did not glow green were considered to be wildtype and were immediately killed in accordance with the regulations
of the University of Washington Institutional Animal Care and Use Committee. On the experimental day GAD67-GFP knock-in mouse pups
were anesthetized by exposing them to air containing isoflurane and
then were rapidly killed by decapitation in accordance with the
regulations of the University of Washington Institutional Animal Care
and Use Committee.
Nonrhythmic medullary slice preparations
We have previously described the methods for obtaining nonrhythmic medullary slices (van Brederode and Berger 2008). Briefly,
J Neurophysiol • VOL
following decapitation a tissue block containing the caudal brain stem
and upper cervical spinal cord were removed from the anesthetized
mouse pup and submerged in ice-cold carbogen-gassed (95% O2-5%
CO2) artificial cerebrospinal fluid (ACSF). The ACSF contained (in
mM): 118 NaCl, 3 KCl, 1 MgCl2, 1 NaH2PO4, 25 NaHCO3, 30
D-glucose, and 1.5 CaCl2. The tissue block was placed on a cutting
platform of a Vibratome (TPI, St. Louis, MO), while this block was
submerged in ice-cold carbogen-gassed ACSF transverse slices (250 –
300 ␮m thick) were cut. Three to four slices were then transferred for
1 h at 37°C to an incubation chamber containing carbogen-gassed
ACSF. Subsequently, slices were held at room temperature until being
transferred to the perfusion-recording chamber. The perfusion-recording chamber was mounted on an upright Zeiss microscope (Axioscope), equipped with both infrared differential interference contrast
(IR-DIC) optics and epifluoresence illumination. The fluorescence
filter set was chosen to reveal, in the absence of infrared illumination,
GFP-containing neurons (QuantaMax-Green filter set, excitation/
emission 450 – 490/510 –560 nm; Omega Optical, Brattleboro, VT).
The slices in the heated perfusion-recording chamber were bathed
at 26 ⫾ 1°C with carbogen-gassed ACSF. For whole cell patch
recordings electrodes were pulled from thin-walled borosilicate glass
to a resistance of 3–7 M⍀ for HMs and 5–10 M⍀ for interneurons.
For current-clamp recordings electrodes were filled with an intracellular solution composed of (in mM): 115 K-gluconate, 25 KCl, 1
MgCl2, 9 NaCl, 10 HEPES, 3 K2-ATP, 1 Na-ATP, and 0.2 K-EGTA.
The pH was adjusted to 7.3 with KOH. In some recordings biocytin
(0.2 wt %) was added to this intracellular solution to allow for post
hoc morphological identification of the cells. A calculated junction
potential of ⫺12 mV was subtracted from the voltage values recorded
in current clamp.
The location of the hypoglossal motor nucleus (nucleus XII) was
determined as described in previous publications from this laboratory
(Viana et al. 1993a). The densely packed multipolar cells that together
form the XII nucleus were identified under high-power optics using
IR-DIC illumination and we selected for recordings large HMs that
were located in the ventrolateral area of the nucleus, an area in which
motoneurons that innervate the genioglossus tongue muscle are located (Krammer et al. 1979). We targeted GFP⫹ interneurons under
epifluorescent illumination. We selected from a large group of GFP⫹
cells only those labeled cells that were located adjacent and just
ventral and lateral to the XII nucleus, in an anatomical area called the
Nucleus of Roller (NR) (Franklin and Paxinos 1997; see Fig. 1). After
verifying that the targeted interneuron was GFP positive we turned off
the fluorescent illumination to prevent cell damage and patched onto
the cell using IR-DIC illumination. Electrodes were advanced onto
visually identified neurons, using positive pressure and, after formation of a gigaohm seal, the cell membrane was ruptured by applying
brief suction to the patch. Whole cell intracellular recordings were
made using an Axoclamp 2B amplifier, a Digidata A/D board and
Clampex software (Molecular Devices, Sunnyvale, CA). Current and
voltage traces were amplified, low-pass filtered at 10 kHz, digitized at
20 kHz, and stored on hard disc. Off-line analysis was done with the
help of Clampfit software (Molecular Devices) and customized procedures in conjunction with the Neuromatics software package (Jason
Rothman) written for IGOR Pro (WaveMetrics). For recording of
intrinsic membrane properties the ACSF contained blockers to minimize background synaptic noise. These included blockers of ␣-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)–mediated
glutamatergic [6,7-dinitroquinoxaline-2,3-dione (DNQX), 10 ␮M],
GABAergic [2-(3-carboxypropyl)-3-amino-6-methoxyphenyl-pyridazinium bromide (SR95531), 0.5 ␮M], and glycinergic [strychnine, 1
␮M] synaptic transmission.
In some recordings we placed a borosilicate glass capillary
stimulating electrode (5- to 10-␮m tip diameter) into the NR to
evoke inhibitory postsynaptic currents (eIPSCs) in the recorded
HMs. Stimuli (40 – 80 V, 0.3-ms pulse duration, 0.5 Hz) were
delivered through a stimulus isolation unit (Grass Instruments),
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tion is presumed to be mediated by GABAergic premotor
neurons that receive excitatory inspiratory drive (Sanchez et al.
2009; Saywell and Feldman 2004). Disruption of GABA synthesis in neonatal mice leads to an irregular respiratory rhythm
characterized by a short inspiratory period and a paucity of
firing during the inspiratory phase in brain stem respiratory
neurons (Kuwana et al. 2003). Blocking GABAA receptors in
the rhythmically active rat brain stem preparation leads to an
increase in inspiratory burst frequency (Bou-Flores and Berger
2001).
HM axons do not possess collaterals (Mosfeldt Laursen and
Rekling 1989; Withington-Wray et al. 1988), suggesting that
there is little or no feedback inhibition. The nature of the
feedforward inhibitory network that controls the output of HMs
to the tongue muscles is not well understood. A major drawback in studying these inhibitory networks has been the difficulty encountered in identifying these neurons in living brain
tissue. The recent development of mice expressing green
fluorescence protein (GFP) under the control of the endogenous GAD67 promotor has made it possible to easily
identify GABAergic neurons in brain tissue (Tamamaki et
al. 2003), including the respiratory brain stem (Kuwana et
al. 2006).
In this study we examined the morphological and electrophysiological properties of GAD67-GFP (GFP⫹) interneurons
located in the NR in both rhythmic and quiescent slices of the
juvenile mouse brain stem. We show that GFP⫹ cells are
tonically active in the rhythmic slice, but this activity is not
modulated by ongoing inspiratory activity. Focal electrical
stimulation of the NR evokes GABAergic postsynaptic currents in HMs. Furthermore, our results show that GFP⫹
interneurons are morphologically and electrophysiologically
heterogeneous, suggesting that they play different roles in
modulating the motor output of HMs.
FIRING PROPERTIES OF BRAIN STEM INTERNEURONS
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driven by Clampex software in the presence of 10 ␮M DNQX and
1 ␮M strychnine. Postsynaptic responses were recorded in voltage
clamp using an Axopatch 200B amplifier (Molecular Devices) at a
holding potential of ⫺70 mV. The intracellular solution for these
recordings was composed of (in mM): 140 CsCl, 1 CaCl2, 4
Cs-BAPTA, 10 HEPES, 5 Mg-ATP, and 10 QX-314. The pH was
adjusted to 7.3 with CsOH.
Rhythmic medullary slice preparations
ON-cell recording of unitary spike activity was made from HMs
and NR GFP⫹ neurons in the rhythmic mouse pup (P5–P9) medullary slice preparation (Smith et al. 1991). We previously described the methods our laboratory uses for obtaining these rhythmic medullary slices (Sebe and Berger 2008; Sebe et al. 2006). In
brief, we cut a single rhythmic slice (600 to 700 ␮m thick) from the
medullary tissue block; the latter was prepared as described earlier
in the case of the nonrhythmic slice preparation. Once cut the
rhythmic slice was immediately placed in the recording-perfusion
chamber described earlier (perfusion rate 4 – 6 ml/min). Over the
course of 30 min extracellular K⫹ was raised to 8 mM in the
carbogen-gassed ACSF (no synaptic blockers added) perfusing
the slice. Chamber temperature was also gradually raised to 27–
28°C before recording commenced. As an index of global rhythmic
inspiratory-phase–related activity in the slice we recorded this
activity using a perfusion fluid–filled glass electrode that had a tip
diameter of 50 –100 ␮m. This field-recording electrode was positioned on the caudal surface of the slice in a region overlying the
ventral respiratory center, just ventral to the compact zone of the
nucleus ambiguus, a region that is known to contain the preBötzinger complex (preBötC). The preBötC generates rhythmic
inspiratory activity in this slice preparation (Smith et al. 1991). In
several experiments we recorded simultaneously using field recording electrodes from both the preBötC and the XII. We found that in these
cases the rhythmic inspiratory activity was linked and occurred simultaneously in both regions. Rhythmic preBötC field activity was sampled at
20 kHz, amplified, and band-pass filtered (10 Hz to 2.4 kHz) using a
CyberAmp 320 amplifier and pCLAMP 10 software (Molecular Devices). Both HMs and GFP⫹ interneurons whose ON-cell spike activity was recorded were identified as described earlier. ON-cell recording
was made using patch recording electrodes described earlier for
J Neurophysiol • VOL
FIG. 1. Nucleus of Roller (NR), located
just ventrolateral to the hypoglossal motor nucleus (XII), contains a high-density of
GAD67-GFP⫹ neurons. A1: confocal microscope image of a nonrhythmic transverse brain
stem slice shows the presence in the NR of a
high density of GAD67-GFP⫹ neurons. The
arrow points to the NR, which is the large
overall cluster of GAD67-GFP⫹ neurons just
ventrolateral to XII (see also Fig. 91 in Franklin and Paxinos 1997). The XII is almost devoid of green fluorescence protein (GFP)–
containing neurons. A2: higher magnification
confocal microscope image of the NR from the
same brain stem slice as in A1. Data in A from
a postnatal day 7 (P7) mouse. B1: infrared
differential interference contrast (IR-DIC) image of a nonrhythmic living transverse brain
stem slice (250 ␮m thick) shows the tip of a
patch recording electrode and the recorded
GAD67-GFP⫹ neuron in the NR. The downward arrow points to the recorded GAD67GFP⫹ neuron. B2: same image as in B1, but in
fluorescence, shows that the recorded neuron
in B1 is GFP positive. Data from a P5 mouse.
Scale is the same as that in B1 and B2, orientation indicators shown (D, dorsal; L, lateral;
M, medial; V, ventral).
GFP⫹ interneurons, except in this case the electrode was filled with
the chamber perfusion fluid. ON-cell spike activity was achieved by
first approaching the visually identified cell with the recording pipette
under positive pressure and then when the pipette tip was in contact
with the somal membrane the pipette’s pressure was made slightly
negative to develop a high-resistance seal. With this seal the electrode
was then able to record well-isolated spikes that were sampled at 20
kHz, amplified, and band-pass filtered (10 Hz to 1.6 kHz) using a
CyberAmp 320 amplifier and pCLAMP 10 software (Molecular Devices). Spikes from these recordings were detected off-line with a
threshold method. Peristimulus time histograms (PSTHs) were constructed by collecting spikes in 50-ms bins for a 2-s time period
coinciding with inspiratory activity recorded extracellularly in the
preBötC. PSTHs were constructed from spikes collected during 15–30
cycles of inspiratory activity.
Subthreshold electrophysiological properties
Long (1-s duration) subthreshold membrane voltage responses to
injected current (I) pulses were used to investigate the passive electrical properties of the neurons. Input resistance (Rn) was calculated
from the steady-state voltage (Vss) response to small (maximum
voltage deflection of between ⫺5 and ⫺10 mV) hyperpolarizing DC
current pulses at rest (Vrest): Rn ⫽ Vss/I. The membrane time constant
(tau) was calculated from exponential curve fits to the initial 50 –100
ms of the same membrane voltage traces. Membrane potential “sag”
was calculated from the ratio of the initial peak voltage (Vpeak)
response and steady-state voltage response (Vss) recorded during the
last 100 ms of the current pulse: sag ratio ⫽ Vpeak/Vss.
Analysis of firing properties
The neurons were classified based on their spiking pattern in
response to DC current pulses. After electrical access to the cell
interior was achieved and membrane potential stabilized we recorded
the membrane voltage responses to a series of depolarizing and
hyperpolarizing DC current steps of 1-s duration at a 10-s interval.
Individual spikes in the voltage trace were detected off-line with a
threshold set at a voltage 20 to 30 mV below the peak amplitude of the
spike. Interspike interval (ISI) and its reciprocal instantaneous firing
frequency ( f ) were calculated from the measured spike threshold
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J.F.M. VAN BREDERODE, Y. YANAGAWA, AND A. J. BERGER
Morphological classification
After completing the intracellular recording protocol, and in those
instances where the intracellular electrodes contained biocytin, the
electrodes were carefully withdrawn from the cell. Slices were fixed
overnight in 4% paraformaldehyde sandwiched between two pieces of
filter paper. Slices were reacted for biocytin with the ABC Elite kit
(Vectastain). The reaction product was visualized with the help of
tablets of diaminobenzidine and H2O2 (Sigma fast kit). Stained tissue
sections were cleared and dehydrated in a graded series of ethanol and
xylene and coverslipped. About 50% of biocytin-filled GFP⫹ cells
and all filled HMs were recovered by this procedure. All filled cells
were photographed with a camera mounted on a microscope. Selected
cells were reconstructed under high power (⫻40 or ⫻100 oil objective) by drawing the outline of the cells on paper with the aid of a
camera lucida. We measured the long- and short-axis diameters of
each cell somata and calculated the soma area from these measurements, assuming an elliptical shape of the soma. We also counted the
number of primary processes coming off the soma.
Statistics
Statistical comparisons were performed using the t-test (paired
comparisons) or ANOVA, with significance set at P ⬍ 0.05. If a
significant difference between group means was found with an
ANOVA, a post hoc Tukey test was performed to determine which
groups differed from each other. Statistical correlations were performed using either linear regression techniques or using Spearman’s
rank correlation. Results are expressed as mean ⫾ SE unless otherwise noted.
RESULTS
Distribution of GAD67-GFP⫹ neurons in the dorsal medulla
Previous studies have shown that between 88 and 97% of
GAD67-GFP⫹ neurons in the knock-in mouse stain for GABA
immunoreactivity (Ono et al. 2005; Tamamaki et al. 2003) and
thus we will consider them to be GABAergic interneurons for
the purpose of this study. GAD67-GFP⫹ neurons were distributed throughout the brain stem, but showed a dense cluster
near the ventrolateral part of the XII nucleus in an area that is
also known as the NR (Fig. 1). Labeled GFP⫹ neurons were
only rarely found inside the XII nucleus. For whole cell
patch-clamp recording we targeted for recording GFP⫹ cells in
the NR close to the border with the XII nucleus under highpower IR-DIC illumination (Fig. 1). GFP fluorescence was
J Neurophysiol • VOL
again confirmed on establishing the whole cell recording configuration.
The quality of the biocytin filling of GFP⫹ cells varied
considerably from cell to cell, with some cells showing only a
relatively vague labeling of the soma and proximal dendrites,
whereas in other cells a more complete filling of the dendritic
tree and, in some cases, the axonal arborizations were
achieved. Biocytin-labeling of intracellularly recorded GFP⫹
cells revealed two major cell types based on the shape and
number of primary processes. The large majority of biocytinlabeled neurons (18 of 21 recovered GFP⫹ cells) had more
than two primary processes emanating from their cell bodies
and were classified as multipolar interneurons (Fig. 2). The
remaining three GFP⫹ neurons had two primary processes
emanating from opposite poles of their cell body and were
classified as fusiform in shape. Multipolar cells had small- to
medium-sized cell bodies (mean soma area ⫽ 111.2 ⫾ 8.7
␮m2, n ⫽ 18) from which originated an average of 4.8 ⫾ 0.4
processes. All three fusiform neurons had cell bodies that were
⬍75 ␮m2 in area (mean soma area ⫽ 63.7 ⫾ 5.8 ␮m2).
Because many of the primary processes were relatively thin it
was often difficult to tell which process was a dendrite and
which was an axon. However, in some well-filled cells we were
able to identify axons with confidence and follow them for
some distance in the slice. The terminal branches of these
processes showed the characteristic small swellings on axon
terminals indicative of putative synaptic contacts (Fig. 2B). In
other cells the axon appeared to be cut off by the slicing
procedure and the terminal end showed a characteristic “bulb,”
where it left the slice (not shown). Although most of the GFP⫹
cells we filled were located with their cell bodies immediately
adjacent to the ventrolateral border of the XII nucleus, their
processes tended to avoid entering the XII nucleus itself. In
about half of the GFP⫹ cells their processes were oriented with
their axes parallel to the capsule that surrounds the XII nucleus
(Fig. 2, B and D). In one such cell we were able to trace its
axon to the contralateral side where it terminated in the capsule
surrounding the XII nucleus on that side (Fig. 2D). Other
GFP⫹ cells were not oriented in a particular orientation relative to the XII nucleus (Fig. 2C). In two GFP⫹ cells the filled
axon ran toward the ventrolateral side of the brain stem slice
away from the XII nucleus (Fig. 2C). In this study we also
filled HMs and these cells had medium- to large-sized multipolar cell bodies (mean soma area ⫽ 292.9 ⫾ 30.7 ␮m2, n ⫽
6, P ⬍ 0.05 vs. GFP⫹ cells). All HMs were located in the
ventrolateral part of the nucleus, in the part that innervates the
genioglossus muscle (Fig. 2A). The morphology of the filled
HMs in this study was similar to what has been described in
previous studies for this type of cell (Nunez-Abades et al.
1994). These cells have extensive dendritic trees that branch
repeatedly within the nucleus and often extend well beyond the
borders of the XII nucleus into the reticular areas adjacent to
the nucleus (Fig. 2A).
Stimulation of the NR evokes inhibitory postsynaptic currents
in HM neurons
It has been known from anatomical studies that the NR
contains neurons that stain for GABA (Aldes et al. 1988).
Electrical stimulation of NR in neonatal mice evokes glycinergic IPSCs in HM neurons (Hulsmann et al. 2000;
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crossings. Stimulus–response curves were created by plotting instantaneous firing frequency against current pulse amplitude ( f–I curves).
For each cell we measured the minimum current necessary to fire at
least one action potential (AP) and this current was defined as the
threshold current (1T). Subsequent current pulses were adjusted such
that their amplitudes were multiples of this threshold current (2T, 3T,
etc.).
The shape of single APs was determined from spikes evoked by
just-suprathreshold current pulses or from rebound APs at the end of
a hyperpolarizing current pulse. Action potential threshold (APth) was
measured from the inflection point at the beginning of the AP
(dVm/dt ⬎ 5 mV/s). Action potential height (APh) was calculated from
the difference between the peak spike voltage and APth. Action
potential half-width (APhw) is reported as the width of the spike at
half-maximal amplitude. The spike afterhyperpolarization (AHP) was
measured as the peak hyperpolarization during the initial 100 ms
following a single spike relative to APth.
FIRING PROPERTIES OF BRAIN STEM INTERNEURONS
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Umemiya and Berger 1995). When we placed a glass stimulating microelectrode into the NR in brain stem slices (two
mice, P7 and P9) and applied brief current pulses we obtained reliable evoked inhibitory postsynaptic currents
(eIPSCs) in HMs (n ⫽ 4 cells) in the presence of 10 ␮M
DNQX and 1 ␮M strychnine (Fig. 3). These eIPSCs were
abolished or severely reduced by bath application of
SR95531 (0.5 ␮M), indicating that they were mediated
chiefly by GABAA receptors. GABAergic IPSCs were also
evoked in HMs by brief puffs of a high potassium solution
(140 mM K⫹) from a glass pipette (1- to 2-␮m tip diameter)
placed near GFP⫹ cells in the NR; these responses disappeared when we moved the puffing pipette away from the
stimulated cells (data not shown). These results, together
with the anatomical data presented earlier, suggest that
GFP⫹ cells inhibit HMs, perhaps through synaptic contacts
onto their distal dendrites. This result is consistent with the
observation in the cat that the NR contains inhibitory neurons that are premotor to HMs (Ono et al. 1998).
J Neurophysiol • VOL
GAD67-GFP⫹ neurons in the NR are tonically active in the
rhythmic slice
To establish the activity pattern of GFP⫹ cells under “in
vivo-like” conditions we recorded spiking activity of GFP⫹
cells in the rhythmic mouse pup (P5–P9) medullary slice
preparation (Sebe and Berger 2008; Smith et al. 1991; van
Brederode and Berger 2008). Rhythmic activity in this slice
preparation was recorded using field electrodes located in the
preBötC. When extracellular K⫹ is raised to 8 mM these slices
generate a regular respiratory-related rhythm recorded as inspiratory burst activity in the preBötC. In these experiments we
did not add synaptic blockers to the bath. In a representative
sample of 13 GFP⫹ cells in the NR we found that all recorded
cells (100% of cells tested) fired spontaneously in rhythmic
slices (Fig. 4). GFP⫹ cells fired spontaneous spikes with a
mean firing frequency of 5.5 ⫾ 1.3 Hz (range: 1.4 –17.5 Hz).
Spontaneous firing was highly regular with a mean coefficient
of variation (CV, calculated from the ratio of the SD and the
mean of the spontaneous firing rate of individual neurons) of
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FIG. 2. Examples of several morphological types
of biocytin-filled mouse brain stem neurons examined in this study. A: photomicrograph of a HM
filled with biocytin through a whole cell intracellular
recording electrode (see METHODS). Slice (250 ␮m
thick, nonrhythmic) was processed for biocytin with
ABC-peroxidase as a whole mount. The cell body
was located in the ventrolateral part of the XII
nucleus (therefore likely a genioglossus motoneuron). The approximate boundary of the nucleus and
the midline of the brain stem are indicated by the
dashed black lines. The black arrow points toward
the axon that, after leaving the nucleus, ran ventrolaterally in the slice toward the ventromedial surface
of the slice. Note the absence of axon collaterals.
The white arrow marks a large dendritic process that
extends well beyond the boundary of the XII nucleus. B: camera lucida reconstruction of a multipolar biocytin-filled GFP⫹ cell located in the NR. The
processes of this cell ran parallel to the boundary of
the XII nucleus (dashed line marks approximate
boundary location). Note the many axonal swellings—presumed synaptic contacts—in the photomicrograph of the terminal axonal arborization of this
cell corresponding to the rectangular area in the
drawing. C: camera lucida reconstruction of a multipolar GFP⫹ cell located in the NR. Arrow points
towards the axon which ran in a ventro-lateral direction in the slice away from the XII nucleus.
D: camera lucida reconstruction of a multipolar
GFP⫹ cell located in the NR. Arrow points to the
cell axon which ran along the border of the XII
nucleus (dashed line), crossed the midline (vertical
dashed line), and ran along the border of the contralateral XII nucleus. D, dorsal; M, medial; CC,
central canal; XII, hypoglossal nucleus.
240
J.F.M. VAN BREDERODE, Y. YANAGAWA, AND A. J. BERGER
A1
A2
XII
XII
D
M
L
50 um
V
A3
SR95531
50 pA
20 ms
FIG. 3. Electrical stimulation of the perihypoglossal region of a nonrhythmic slice containing GAD67-GFP⫹ neurons results in ␥-aminobutyric acid type A
(GABAA) receptor-mediated synaptic currents in a hypoglossal motoneuron (HM). A1: IR-DIC image of slice shows electrical stimulation electrode in the
perihypoglossal area just ventrolateral to the hypoglossal motor nucleus and the simultaneously recorded HM. Tip of the stimulating electrode indicated by the
downward arrow, tip of patch recording electrode, and the recorded HM indicated by the upward arrow. A2: same image as that in A1, but in fluorescence showing
the cluster of GAD67-GFP⫹ neurons in the NR. Upward and downward arrows in same position as in A1 and A2. Scale bar same in A1 and A2, orientation
indicator shown (D, dorsal; L, lateral; M, medial; V, ventral; XII, hypoglossal motor nucleus). A3: average traces of GABAA receptor-mediated evoked IPSCs
before and during bath application of SR95531 (0.5 ␮M). Experiments were performed in the presence of DNQX (10 ␮M) and strychnine (1 ␮M). Additional
blockade of GABAA receptors with SR95531 almost completely abolished the evoked response. Data shown were obtained from the stimulating and recording
electrodes shown in A and the responses shown are average responses to 10 consecutive stimuli in control and during SR95531 application; data taken from a
P9 mouse. DNQX, 6,7-dinitroquinoxaline-2,3-dione; IPSC, inhibitory postsynaptic current; SR95531, 2-(3-carboxypropyl)-3-amino-6-methoxyphenyl-pyridazinium bromide.
0.28 ⫾ 0.05 and was maintained with little variation in mean
spontaneous rate for recording periods ⱕ30 min (data not
shown). Importantly, this ongoing tonic activity was not modulated by coincident inspiratory burst activity in the preBötC
(Fig. 4B). The spontaneous firing rate of GFP⫹ cells in the NR
was unaffected by adding a cocktail of blockers of glutamatergic (10 ␮M DNQX in combination with 10 ␮M 2-amino5-phosphonopentanoic acid [AP-5]), GABAergic (0.5 ␮M
SR95531), and glycinergic (1 ␮M strychnine) neurotransmission to the bath (n ⫽ 5 cells, P ⬍ 0.05), suggesting that
autorhythmicity is an intrinsic property in these cells, independent of synaptic drive. HMs, in contrast, were generally not
tonically active in this preparation, but instead showed spiking
activity only during and coincident with inspiratory burst
activity in the preBötC (Fig. 4A).
Comparison of the electrophysiological properties of
GAD67-GFP⫹ and HM neurons
We obtained stable whole cell intracellular recordings from
a total of 43 GAD67-GFP⫹ neurons in the NR. All whole cell
intracellular recordings of intrinsic membrane properties were
obtained with blockers of glutamatergic, GABAergic, and
glycinergic neurotransmission present in the bath (nonrhythmic
slice; see METHODS). We only report on GFP⫹ neurons that had
overshooting (⬎0 mV) action potentials and input resistances
⬎200 M⍀. In the nonrhythmic slice (P5–P15 mouse pups),
J Neurophysiol • VOL
slightly more than half of the GFP⫹ neurons (24 of 43) fired
spontaneous spikes at rest at a rate of 5.9 ⫾ 1.3 Hz (mean ⫾
SE, n ⫽ 12). For the analysis of firing properties (see following
text) it was necessary to inject a small amount of DC hyperpolarizing current into these cells to prevent spontaneous
spiking. The amount of negative current was adjusted in this
group of cells such that the membrane voltage was just below
the threshold for spontaneous spiking (average hyperpolarizing
injected current was ⫺17.0 ⫾ 2.26 pA). Mean resting membrane potential of quiescent and spontaneously active GAD67GFP⫹ cells, after injection of hyperpolarizing holding current,
was not significantly different between these two groups
(⫺69.2 vs. ⫺64.5 mV, respectively, P ⬎ 0.05). Mean input
resistance (Rn) was higher in spontaneously active cells than
that in quiescent GFP⫹ cells (973 vs. 523 M⍀, respectively,
P ⬍ 0.05). In addition, action potential height and membrane
time constant were not significantly different between these
two groups, suggesting that spontaneous firing at rest was not
due to increased leakage current resulting from cell damage or
other factors leading to unhealthy or unstable cells. We believe
that the spontaneous activity in GFP⫹ cells in the NR in the
nonrhythmic slice is a reflection of the spontaneous firing that
we found with ON-cell recordings in the rhythmic slice as
described earlier. For the purpose of analyzing the firing
properties of GFP⫹ cells we combined the data from spontaneously active and quiescent cells. To compare GFP⫹ cell
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Control
FIRING PROPERTIES OF BRAIN STEM INTERNEURONS
241
GFP+
HM
B1
A1
0.2 mV
500 ms
A2
B2
0.2 mV
500 ms
B3
Burst#
25
16
1
1
A4
-500
0
500
1000
1500
B4
0
0
500
500
1000
1500
1000
1500
2000
20
Spikes / bin
Spikes / bin
20
-500
15
10
5
0
15
10
5
0
0
500
1000
1500
2000
Time (ms)
Time (ms)
firing patterns in this study to those of HMs (van Brederode
and Berger 2008; Viana et al. 1995) we also obtained recordings from seven HMs located in the genioglossus division of
the XII nucleus. None of the recorded HMs were spontaneously active at rest in the nonrhythmic slice.
Firing patterns in response to depolarizing current steps
We found that GFP⫹ neurons in the brain stem are electrophysiologically heterogeneous. We were able to distinguish
two different basic firing patterns—tonic and phasic spiking—
based on the responses to long (1-s) step depolarizing current
stimulation. These different firing patterns were most obvious
when cells were stimulated with just-suprathreshold current
pulses at resting membrane potential. Examples of these two
basic firing patterns are shown in Fig. 5A. Tonic cells (n ⫽ 36)
fired a regular train of action potentials in response to positive
current pulses (Fig. 5A1). At just-suprathreshold current amplitude (threshold current or 1T amplitude; see METHODS) these
cells fired multiple spikes at low frequency throughout the
J Neurophysiol • VOL
duration of the pulse. Tonic cells could be further subdivided
into three subcategories based on their firing patterns (Fig. 6).
Tonic, adapting cells (n ⫽ 17) showed a gradual lengthening
of ISIs with successive spikes in the train (i.e., spike-frequency
adaptation, Fig. 6A). Tonic, nonadapting cells (n ⫽ 10) showed
little or no such spike-frequency adaptation; spike intervals did
not vary systematically from the beginning to the end of the
train (Fig. 6C). Tonic, accelerating cells (n ⫽ 9) showed a
decrease in ISI (or an increase in instantaneous spike frequency) with successive spikes in the train (Fig. 6B). A
minority of cells in this latter group (4 of 9) had a long first ISI
and relatively long delay to the first action potential in the train
at just-suprathreshold stimulus current (Fig. 6B). To quantify
differences in firing types among tonic GFP⫹ interneurons we
made frequency–time ( f–t) plots based on the response of
neurons to DC current stimulation. The amount of spikefrequency adaptation was dependent on the amplitude of the
current pulse and membrane depolarization. To permit a classification of neurons based on f–t relationships we calculated
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A3
Burst#
FIG. 4. Examples of the spiking patterns of an HM (A)
and NR GFP⫹ neuron (B) recorded in rhythmically active
mouse brain stem slices. A1 and B1, top traces: integrated
electrical activity recorded with a field electrode placed in
the ventral medulla in the vicinity of the pre-Bötzinger
complex (preBötC). Raw field recordings were rectified,
integrated, and aligned at the start of each inspiratory burst
and averaged. A2 and B2: examples of the spikes recorded
simultaneously with a cell-attached whole cell recording
electrode during a single inspiratory burst. GFP⫹ cell was
tonically active at a mean rate of 12.7 Hz (with a coefficient
of variation [CV] of 0.16), whereas the HM fired only
during the inspiratory burst. A3 and B3: raster plots for the
spiking activity during 16 (left) and 25 (right) inspiratory
cycles in the 2 neurons. The start of each inspiratory burst
was aligned at 0 ms. Spike time occurrences in individual
bursts were measured over a time interval starting 500 ms
before the start of the burst for a total duration of 2 s. A4 and
B4: peristimulus time histograms calculated for the raster
plots shown above. Bin width: 50 ms. Note the prominent
modulation of firing rate of the HM during the burst and the
absence of inspiratory-burst modulated activity in the NR
GFP⫹ cell.
242
J.F.M. VAN BREDERODE, Y. YANAGAWA, AND A. J. BERGER
Tonic GFP+
A1
-60
Phasic GFP+
HM
A2
B
-72
-74
20 mV
500 ms
100 pA
500 pA
20 mV
50 ms
the adaptation ratio (first spike interval frequency/last spike
interval frequency) for a stimulus of twofold the threshold
amplitude (2T) to elicit repetitive firing at resting membrane
potential. This adaptation ratio was used to classify tonic
GFP⫹ interneurons (Table 1). Tonic interneurons that had
adaptation ratios ⬍0.90 were classified as tonic, accelerating
cells, neurons with ratios between 0.90 and 1.10 as nonadapting cells, and neurons with ratios ⬎1.1 as adapting cells.
Phasic GFP⫹ cells (n ⫽ 7) did not fire throughout the pulse
when they were stimulated with 1-s depolarizing current steps
at just-suprathreshold current strength. Phasic GFP⫹ cells
could be subdivided into cells that fired single spikes only
(single spike cells; n ⫽ 2, Fig. 5A2), fired a brief burst of spikes
(⬍4) at the beginning of the pulse (single burst cells; n ⫽ 2,
Fig. 7A), or fired multiple spikes after a long delay to the first
spike in the train (delayed onset cells; n ⫽ 3, Fig. 7B).
Spontaneous oscillations of the membrane potential near spike
threshold were common in these cells (Fig. 7, A and B). The
firing characteristics of phasic GFP⫹ cells were dependent on
stimulus strength and membrane depolarization. For instance,
delayed onset were able to fire throughout the pulse after only
a short delay when the stimulus amplitude was increased
beyond threshold current (Fig. 7B) and single spike cells could
fire more than one spike when they were depolarized to just
below spike threshold (data not shown). Delayed onset cells
often showed irregular firing (data not shown). Other studies
have found that these subtypes of interneurons (single spike,
single burst, or delayed onset) constitute separate cell classes
(Prescott and De Koninck 2002), although we found too few of
these subtypes to classify them into separate categories and, for
the purpose of this study, they were lumped together in one
group that we term phasic cells based on the distinction that
they do not fire a regular train of spikes throughout the pulse.
Furthermore, classification of these particular cell types was
complicated by the observation that these firing patterns were
J Neurophysiol • VOL
also the most sensitive to changes in membrane potential and
stimulus current.
HMs (n ⫽ 7) all fired a regular train of action potentials in
response to depolarizing current pulses (Figs. 5B and 7C),
similar to what has been described in HMs in the rodent brain
stem in previous studies (Nunez-Abades et al. 1993; van
Brederode and Berger 2008; Viana et al. 1995). HMs in this
study showed either little or no frequency adaptation or frequency acceleration due to a long first ISI. Previous studies
from our laboratory have shown that the majority of HMs in
rats show frequency adaptation during the first postnatal week,
whereas an accelerating firing pattern is more common during
the second postnatal week (Viana et al. 1995). Consistent with
this observation in rats we found that HMs that showed
acceleration were recorded in slightly older mice (n ⫽ 3
neurons, mean age ⫽ 11.3 days) than HMs that showed
adaptation (n ⫽ 4 neurons, mean age ⫽ 8.7 days). Three HMs
showed a long delay to the first spike at just-suprathreshold
current (Fig. 7C1). f–I curves for the first and last intervals in
the spike trains of HMs were linear (Fig. 7C2), as previously
described for genioglossus HMs in earlier studies (NunezAbades et al. 1993; Viana et al. 1995).
Passive properties of the four groups of interneurons and the
group of HMs are given in Table 1. There was no significant
animal age difference between different types of tonic GFP⫹
cells (ANOVA, P ⬎ 0.05) or between tonic and phasic GFP⫹
cells (t-test, P ⬎ 0.05), but HM recordings were obtained from
slightly older animals than recordings from GFP⫹ cells (mean
postnatal ages 10.1 vs. 7.2 days, respectively).
Neurons in all cell groups showed a variable amount of
depolarizing sag in response to hyperpolarizing current pulses
(Fig. 5, Table 1). Sag ratios, calculated from the membrane
voltage response to 20 to 30 mV hyperpolarizing current pulses
(see METHODS), were not significantly different among neuron
groups. Sag ratio in GFP⫹ interneurons was not correlated
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FIG. 5. Examples of the electrophysiological properties
of the 2 main classes of GFP⫹ neurons (tonic, A1; phasic,
A2) and HMs (B). Shown are membrane voltage responses
(resting membrane potential indicated in the top traces) to
2 depolarizing steps of different amplitude (just-suprathreshold voltage response is shown in the middle) and a
hyperpolarizing current step recorded at resting membrane
potential (current pulses shown below voltage traces).
Tonic cells fired a regular train of action potentials (APs) in
response to depolarizing current pulses (A1). These cells
often showed rebound APs at the end of a hyperpolarizing
current pulse (bottom voltage trace). Note the large depolarizing “sag” in the membrane potential trace in response
to the hyperpolarizing current step. Single APs (bottom
trace; note expanded timescale) were followed by a relatively slow single hyperpolarizing afterpotential (AHP).
Phasic cells (A2) fired a single AP or only a few APs (⬍4)
at the start of the pulse. Single APs (bottom) were followed
by a fast AHP, which slowly decayed back to threshold.
HMs (B) fired a regular train of APs in response to depolarizing current pulses. Single APs were usually followed
by 3 afterpotentials, a fast AHP, an afterdepolarization
(ADP), and a medium AHP. All the data in this and the
remaining figures were obtained from nonrhythmic slices in
the presence of glutamatergic, GABAergic, and glycinergic
antagonists (see METHODS).
FIRING PROPERTIES OF BRAIN STEM INTERNEURONS
A1
B1
Adapting
C1
Accelerating
-72
-69
243
Non-adapting
-70
20 mV
500 ms
B2
Firing Frequency (Hz)
A2
C2
30
30
30
20
20
20
1st
avg
last
10
10
10
0
0
0
20
40
60
20
40
60
80 100
Stim Current (pA)
B3
Firing Frequency (Hz)
A3
0
0
80 100
0
10
20
15
15
10
10
5
0
120
25
30
20
80
C3
20
4T
2T
1T
40
5
0
200 400 600 800 1000
0
200 400 600 800 1000
200 400 600 800 1000
Time (ms)
FIG. 6. Examples of the firing patterns in response to step-current pulses in a tonic adapting (column A), tonic accelerating (column B), and a tonic
nonadapting (column C) GFP⫹ interneuron. Top panel (A1–C1): for each cell membrane voltage responses to 1,000-ms-long depolarizing current pulses at 3
different amplitudes are shown. Current amplitude increases from bottom to top. The smallest current pulse that resulted in repetitive firing is the threshold current
(1T). Resting membrane potential is indicated in the top trace of each panel. Middle panel (A2–C2): relationship between instantaneous firing frequency and
current amplitude ( f–I plot) for the cells shown in the top panel. Plotted are the firing frequency for the first spike interval in the train (first; filled squares), last
interval (last; open squares), and average frequency (avg; pluses). Graphs for first, last, and average frequencies and current amplitudes were fitted with straight
lines and the slope of these lines was used to quantify f–I relationships with current amplitude expressed relative to the threshold current (1T). Bottom panel
(A3–C3): relationship between the instantaneous firing frequency and the time from the start of the pulse ( f–t plot) for the cells shown in the top panel. For each
cell the f–t plots at 3 different current amplitudes (expressed as multiples of the threshold current) are shown corresponding to the 3 traces in the top panel. Note
the gradual lengthening of interspike intervals (ISIs) in the adapting cell in A. The cell in B increases its firing rate rapidly for the first few intervals and more
gradually later in the train. Spike intervals remain virtually constant in the nonadapting cell in C.
with animal age (correlation coefficient r ⫽ 0.06, P ⬎ 0.05).
Steady-state input resistance (Rn) was significantly larger in
interneurons than that in HMs (t-test, P ⬍ 0.05, see Table 1).
Phasic cells had a lower Rn than that in the other types of
interneurons (Tukey test, P ⬍ 0.05). Whole cell membrane
time constant (tau) was significantly longer in interneurons
than that in HMs (t-test, P ⬍ 0.05, see Table 1). Phasic cells
had a shorter tau than that of the other types of interneurons
(P ⬍ 0.05) and their tau was not significantly different from the
tau of HMs (Tukey test, P ⬎ 0.05). Whole cell capacitance (C)
was substantially larger in HMs than that in interneurons
(t-test, P ⬍ 0.05), which is a reflection of their much larger cell
J Neurophysiol • VOL
size. Whole cell capacitance was not different among different
types of GFP⫹ interneurons (Table 1; ANOVA, P ⬎ 0.05).
In addition to differences in firing patterns and passive
membrane properties we found that the subclasses of GFP⫹
cells and HMs differed based on the shape and time course of
single action potentials (Table 1). Action potential threshold
and peak height were not different between HMs and interneurons or among interneurons, but spike half-width was shorter in
HMs than that in interneurons (Table 1; t-test and ANOVA,
P ⬍ 0.05). Tonic, adapting neurons had significantly wider
spikes than those of the other groups of GFP⫹ interneurons
(Table 1; Tukey test, P ⬍ 0.05). In addition to differences in
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200 pA
244
J.F.M. VAN BREDERODE, Y. YANAGAWA, AND A. J. BERGER
TABLE
1 Intrinsic membrane properties of GAD67-GFP⫹ interneurons and hypoglossal motoneurons
Cell Type
n
Tau, ms
Rn, m⍀
C, pF
Sag-ratio
Vrest
62 ⫾ 6
50 ⫾ 7
62 ⫾ 7
42 ⫾ 6
117 ⫾ 28*
⬍0.05
1.17 ⫾ 0.05
1.30 ⫾ 0.09
1.13 ⫾ 0.03
1.10 ⫾ 0.02
1.31 ⫾ 0.08
⬎0.05
⫺69.0 ⫾ 2.1
⫺69.3 ⫾ 2.0
⫺72.1 ⫾ 2.7
⫺68.3 ⫾ 2.2
⫺75.4 ⫾ 1.7
⬎0.05
APhw, mV
AHP, mV
tAHP, ms
A. Passive properties
Interneurons
Tonic, adapting
Tonic, nonadapting
Tonic, accelerating
Phasic
HMs
ANOVA, P value
Cell Type
17
10
9
7
7
888 ⫾ 130
788 ⫾ 146
808 ⫾ 81
375 ⫾ 68†
57 ⫾ 130*
⬍0.05
n
APth, mV
47 ⫾ 5
57 ⫾ 12
47 ⫾ 5
15 ⫾ 3†
6 ⫾ 1*
⬍0.05
APh, ms
B. Action potential properties
17
10
9
7
7
⫺51.6 ⫾ 2.1
⫺54.0 ⫾ 1.4
⫺50.3 ⫾ 2.5
⫺52.2 ⫾ 1.3
⫺54.3 ⫾ 2.2
⬎0.05
56.0 ⫾ 4.0
58.7 ⫾ 3.3
59.3 ⫾ 5.1
50.7 ⫾ 2.6
62.3 ⫾ 4.9
⬎0.05
1.76 ⫾ 0.14
1.28 ⫾ 0.10
1.15 ⫾ 0.08
1.39 ⫾ 0.17
0.81 ⫾ 0.05*
⬍0.05
⫺20.6 ⫾ 0.8
⫺22.3 ⫾ 1.1
⫺23.1 ⫾ 2.1
⫺17.1 ⫾ 2.0†
⫺16.2 ⫾ 1.4*
⬍0.05
44.5 ⫾ 5.93
33.6 ⫾ 2.7
28.0 ⫾ 3.6
11.0 ⫾ 1.8†
9.5 ⫾ 3.1*
⬍0.05
Cell Type
n
Adap-ratio
Ith, pA
fth, Hz
fI slope, Hz
fL slope, Hz
4.4 ⫾ 0.5
5.0 ⫾ 0.5
4.9 ⫾ 0.7
7.8 ⫾ 2.3†
8.8 ⫾ 1.2*
⬍0.05
9.5 ⫾ 1.7
4.1 ⫾ 0.6
4.1 ⫾ 0.3
12.8 ⫾ 3.4
15.3 ⫾ 3.7*
⬍0.05
4.2 ⫾ 0.8
2.8 ⫾ 0.5
3.6 ⫾ 0.4
8.0 ⫾ 1.9
17.4 ⫾ 3.3*
⬍0.05
C. Firing properties
Interneurons
Tonic, adapting
Tonic, nonadapting
Tonic, accelerating
Phasic
HMs
ANOVA, P value
17
10
9
7
7
1.31 ⫾ 0.04
1.01 ⫾ 0.02
0.83 ⫾ 0.03
NA
0.86 ⫾ 0.10*
⬍0.05
22.5 ⫾ 4.2
11.3 ⫾ 1.7
16.3 ⫾ 3.4
42.9 ⫾ 9.2†
367.9 ⫾ 77.8*
⬍0.05
Values are means ⫾ SE. Rn, input resistance; tau, membrane time constant; C, total cell capacitance; Sag, sag-ratio calculated from: Vpeak/Vss (see METHODS);
Vrest, resting membrane potential; APth, action potential threshold; APh, action potential height; APhw, action potential half-width; AHP, magnitude of the
maximum spike afterhyperpolarization; tAHP, time to the peak spike afterhyperpolarization; Adap-ratio, frequency adaptation ratio calculated from the firing
response to square current pulses (see METHODS); Ith, minimum or threshold current amplitude needed to elicit repetitive firing; fth, threshold frequency for
repetitive firing; f1slope, slope of the relationship between the instantaneous frequency of the first spike interval in the train and the amplitude of the injected
current pulse normalized by expressing current as a multiple of the threshold current (threshold current ⫽ 1); fL slope, slope of the normalized f–I relationship
for the last spike interval in the train. ANOVA was performed between all five groups of cells. Significant differences between groups are discussed in the
RESULTS. *Significant difference between interneurons and HMs (t-test, P ⬍ 0.05). †Significant difference between phasic and tonic interneurons (Tukey test,
P ⬍ 0.05).
spike shape we found that neurons in each class could be
distinguished based on the shape and time course of their spike
afterpotentials (Fig. 5, Table 1). In all phasic GFP⫹ cells (n ⫽
7) spikes were followed by a single fast afterhyperpolarization
(AHP) that decayed gradually back to the threshold potential
(Fig. 5A2). In the majority of tonic GFP⫹ cells spikes were
followed by a single gradual medium duration AHP (Fig. 5A1;
seen in 21 of 36 cells). Spikes in the other tonic GFP⫹ cells
were characterized by afterpotentials composed of an afterdepolarization (ADP; sometimes visible only as a slight “hump”
in the membrane trajectory following a spike) followed by an
AHP (n ⫽ 14, data not shown), whereas in one tonic cell spike
afterpotentials were triphasic consisting of fAHP, ADP, and
mAHP (data not shown; see Schwindt et al. 1988). The peak
value of the AHP was larger and the time to the peak of the
AHP (tAHP) was slower in tonic interneurons than that in phasic
interneurons or HMs (Table 1; Tukey test, P ⬍ 0.05). Triphasic
afterpotentials (fAHP, ADP, and mAHP) were common (5 of
7 cells) in HMs (Fig. 5B; see also Viana et al. 1993b).
When we measured the lowest firing frequency in response
to the smallest long depolarizing current pulse that resulted in
repetitive firing (threshold frequency or fth) we found lower
values for fth in tonic cells than in phasic cells or HMs (Table
J Neurophysiol • VOL
1, Tukey test, P ⬍ 0.05). We plotted the value of the instantaneous firing frequency for the first interval ( f1) and the last
interval ( fL) in the train against current amplitude (expressed
as multiple of the threshold current or 1T; see METHODS) to
construct f–I relationships. For small to moderate current
amplitudes these f–I relationships were linear or nearly so
(Figs. 6 and 7). The slope of the f1–I relationships (which
presumably reflects the nonadapted state of the cell) was
steeper in tonic adapting neurons than in tonic nonadapting or
accelerating cells (Table 1; Tukey test, P ⬍ 0.05). In the
adapted or steady state there was no difference between the
slopes of the fL–I relationship between the three classes of
tonic cells (Tukey test, P ⬎ 0.05). The slope of the f1–I
relationship in phasic cells was steeper than that in tonic cells,
but this difference was significant only versus tonic nonadapting and accelerating cells (Table 1, Tukey test, P ⬍ 0.05). The
slopes of the f1–I and fL–I relationships were the steepest in
HMs and significantly larger in HMs than in tonic interneurons
(but not different from phasic interneurons). Beyond current
strengths of 2T most tonic nonadapting cells began to show
mild spike-frequency adaptation, especially in the first few
intervals, and this is reflected in the smaller slope of the fL–I
slope compared with the f1–I slope (Table 1, t-test, P ⬍ 0.05).
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Interneurons
Tonic, adapting
Tonic, nonadapting
Tonic, accelerating
Phasic
HMs
ANOVA, P value
FIRING PROPERTIES OF BRAIN STEM INTERNEURONS
A1
B1
Phasic GFP+
-62
Delayed-onset GFP+
245
C1
-62
HM
-74
20 mV
500 ms
500 pA
200 pA
Firing Frequency (Hz)
B2
30
30
40
1st
avg
last
20
20
30
20
10
10
10
0
0
0
0
10
20
30
40
50
A3
Firing Frequency (Hz)
C2
50
0
B3
50
100
0
150
100 200 300 400 500
C3
Stim Current (pA)
50
30
30
40
20
2.5T
1.8T
1T
10
0
30
20
20
7T
3T
1T
10
0
200 400 600 800 1000
1.7T
1.3T
1T
10
0
200 400 600 800 1000
200 400 600 800 1000
Time (ms)
FIG. 7. Examples of the firing patterns in response to step current pulses for a phasic interneuron (A), a delayed onset interneuron (B), and an HM (C). Top
panel (A1–C1): for each cell membrane voltage responses to 1,000-ms-long depolarizing current pulses at 3 different amplitudes are shown. Current amplitude
increases from bottom to top. The smallest current pulse that resulted in repetitive firing is the threshold current (1T). Resting membrane potential is indicated
in the top trace of each panel. Middle panel (A2–C2): relationship between instantaneous firing frequency and current amplitude ( f–I plot) for the cells shown
in the top panel. Plotted are the firing frequency for the first spike interval in the train (first; filled squares), last interval (last; open squares), and average frequency
(avg; pluses). For the phasic cell only the first spike interval frequency is shown. Graphs for first, last, and average frequencies and current amplitudes were fitted
with straight lines. Bottom panel (A3–C3): relationship between the instantaneous firing frequency and the time from the start of the pulse ( f–t plot) for the cells
shown in the top panel. For each cell the f–t plots at 3 different current amplitudes (expressed as multiples of the threshold current) are shown corresponding
to the 3 traces in the top panel. Note the long delay to the first spike in the train at 1T current amplitude for the delayed onset cell and the HM cell. These 2
cell types also showed relatively little spike frequency adaptation.
In HMs the f1–I and fL–I relationships did not differ (t-test, P ⬎
0.05), reflecting the lack of adaptation even at relatively high
stimulus strengths in these cells. In tonic cells that had a spike
ADP the f1–I relationships deviated from linear at high current
strengths when these cells started to fire initial spike “doublets”
(data not shown).
cell-attached and whole cell recordings, suggesting that these
cells possess intrinsic spontaneous activity. Whole cell intracellular recording and filling of GFP⫹ interneurons indicate
that they are electrophysiologically and morphologically heterogeneous.
Morphology of GFP⫹ cells
DISCUSSION
Summary of results
In this study we describe the morphological and electrophysiological properties of a population of GABAergic interneurons in the NR. We show that stimulation of this area in the
brain stem evokes GABAergic postsynaptic responses in HMs
in the adjacent XII nucleus. A large proportion of GFP⫹
interneurons in the NR were spontaneously active both in
J Neurophysiol • VOL
We found a dense cluster of GAD67-positive, small- to
medium-sized cells located in the mouse NR, similar to what
has been described in the rat NR (Tanaka et al. 2003). The
somadendritic morphology of GFP⫹ interneurons in the NR
was quite variable, but we found that the axonal and dendritic
trees of these cells were often restricted to a narrow band
surrounding the ventrolateral side of the XII nucleus. In addition, the main orientation of the cell processes often ran
parallel to the border of the XII nucleus. The functional
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A2
246
J.F.M. VAN BREDERODE, Y. YANAGAWA, AND A. J. BERGER
Firing types of GFP⫹ cells and functional consequences for
inhibitory circuitry
GAD67-GFP⫹ neurons in the NR in this study share electrophysiological and morphological characteristics with
GAD67-GFP⫹ neurons located in the preBötC (Kuwana et al.
2006) and the inferior colliculus (Ono et al. 2005). A subpopulation of GAD67-GFP⫹ cells in the preBötC shows spontaneous tonic spiking (Kuwana et al. 2006), similar to what we find
in the NR. GAD67-GFP⫹ cells in the inferior colliculus and
preBötC include regular spiking cells with mild frequency
adaptation, similar to our tonic adapting cells. In addition, Ono
et al. (2005) also found GAD67-GFP⫹ cells with a long delay
to the first spike (i.e., delayed onset cells), GFP⫹ cells with a
long first spike interval combined with frequency acceleration,
and phasic cells similar to these cell types described in our
study. Similar firing types are also found in spinal cord interneurons (Prescott and De Koninck 2002), suggesting that
certain circuit dynamics are similar across different brain stem
and spinal cord regions and that they require similar types of
interneurons. In the spinal cord these different firing types are
associated with distinct morphological properties (Prescott and
De Koninck 2002), but in our study we did not find such a
correlation. This was perhaps attributable to the low percentage
of phasic cells that we encountered in our study. Tonic inhibitory cells in the spinal cord express a persistent sodium current
(INaP) and a persistent calcium current (ICaP). The intrinsic
properties make these cells optimally suited to encode stimulus
J Neurophysiol • VOL
amplitude and function as integrators of synaptic input (Prescott and De Koninck 2002, 2005). The intrinsic properties of
phasic cells in contrast were such that they functioned optimally as coincidence detectors (Prescott and De Koninck
2002). In the companion paper we show that a similar distinction can be applied to tonic and phasic GABAergic interneurons in the NR based on their responses to time-varying stimuli
(van Brederode and Berger 2011).
The firing properties of GAD67-GFP⫹ interneurons in the
NR will influence how they encode synaptic input and what
type of stimulus is optimally suited to excite them. For instance, single spike cells and cells that fire only a few spikes at
the beginning of the stimulus stopped firing well before the end
of the current pulse and thus prefer short, nonsummating
synaptic inputs. Single spike cells are also unable to encode
stimulus intensity by changing their spike frequency. Tonic
cells can encode stimulus intensity by linearly varying their
firing frequency, depending on input amplitude and their ability to fire trains of action potentials allows them to integrate
synaptic inputs over long time periods. If one combines these
different firing patterns with the observed differences in threshold frequency and membrane time constant among GFP⫹
interneurons in the NR, it is not difficult to imagine that one
can selectively activate a subgroup of interneurons in the NR
based on the temporal pattern and intensity of the stimulus. It
remains to be determined whether the different NR inhibitory
interneuron cell types are involved in encoding different types
of oropharyngeal behaviors and whether their input– output
properties are matched to the specific task that each plays in
these behaviors.
A subclass of GFP⫹ cells and a majority of HMs in this
study showed spike-frequency acceleration in response to DC
current step. This property has been described previously in
HMs (Viana et al. 1995) and in neocortical pyramidal cells
(Miller et al. 2008; Spain et al. 1991). In neocortical pyramidal
cells spike-frequency acceleration is mediated by an ID-like
potassium current that is sensitive to blockade by dendrotoxin-I
and is associated with Kv1 subunit expression (Miller et al.
2008). Spike-frequency acceleration in neocortical pyramidal
cells was accompanied by a relatively long delay to the first
spike in the train and we found a similar association between
the two properties in the group of tonic GFP⫹ cells that
showed spike-frequency acceleration. The function of spikefrequency acceleration is not known, but it might enhance the
gain and/or synchronization of neurons that express this property (Miller et al. 2008).
Tonic activity and its consequences
We showed that GFP⫹ neurons in the NR display spontaneous tonic firing that is not modulated by inspiratory synaptic
drive in the rhythmically active in vitro slice preparation. This
tonic spiking activity of GFP⫹ neurons was not dependent on
synaptic drive and was confirmed in intracellular recordings in
the nonrhythmic slice where fast synaptic transmission was
blocked, suggesting that it is due to genuine autorhythmicity.
We have not investigated the ionic mechanisms behind autorhythmicity in GFP⫹ NR neurons in this study. Autorhythmicity in other types of neurons is mediated by a combination of
pacemaker currents responsible for interspike membrane depolarization and a “reset” mechanism composed of spike afte-
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consequences of this particular anatomical arrangement remain
to be established. The axonal anatomy of filled GFP⫹ cells in
this study suggests that synaptic contacts are made with other
neuronal structures in a narrow band surrounding the ventrolateral part of the XII nucleus on both the ipsilateral side and
the contralateral side. Axonal branching patterns suggest that
GFP⫹ cells in the NR make contacts with structures close to
their own cell bodies and with remote structures. Our study did
not identify those targets, nor did we establish whether the
axonal boutons that we observed were actual synaptic contacts.
However, if at least some of these boutons are synaptic connections between GFP⫹ cells and HMs, then this implies that
these synapses are located on HM dendrites that extend beyond
the boundaries of the XII nucleus. Dendrites that extend well
beyond the border of the XII nucleus and into the lateral
reticular formation have been described in the rodent brain
stem (Altschuler et al. 1994; Nunez-Abades et al. 1994; TarrasWahlberg and Rekling 2009) and we confirm this observation.
These distal dendrites are thus a possible location for synaptic
contacts between the axon terminals of GFP⫹ cells in the NR
and HMs.
We saw no filled interneuron axons enter the XII nucleus,
consistent with a study in the monkey that found that no
GABA-positive axonal pathways from outside the XII nucleus
entered the nucleus itself; instead, the inhibitory synaptic
terminals on HMs originated from GABAergic interneurons
located inside the XII nucleus (Takasu et al. 1987). Rarely did
we observe GAD67-GFP⫹ neurons with cell bodies located
inside the XII nucleus, suggesting that there might be two inhibitory circuits impinging on HMs, one originating from inside the
nucleus and the other from perihypoglossal regions in the reticular
formation, including the NR.
FIRING PROPERTIES OF BRAIN STEM INTERNEURONS
Functional significance
One of the roles of the genioglossus muscle is to maintain
airway patency. There is increasing evidence that a disruption
of reflex pathways involving the genioglossus muscle contributes significantly to obstructive sleep apnea (Wheatley et al.
1993). In vivo studies have shown that hypoglossal motoneurons are tonically inhibited by GABA and that antagonists of
this inhibitory neurotransmitter increase the level of spiking
activity in HMs (Liu et al. 2003; Morrison et al. 2003), but this
was not observed for glycine receptor antagonists (Morrison et
al. 2002). However, despite this powerful tonic synaptic inhibition no role of GABAergic inhibition was found in the
suppression of genioglossal activity during rapid eye movement (REM) sleep (Morrison et al. 2003), but others have
found that during cholinergically induced REM sleep a powerful glycinergic inhibitory premotor system suppresses hypoJ Neurophysiol • VOL
glossal motoneurons (Yamuy et al. 1999). Negative pressure in
the upper airway causes a reflex increase in genioglossus
activity, which is likely caused by a release from tonic inhibition of HMs mediated by inhibitory premotoneurons in the
reticular formation (Chamberlin et al. 2007). Thus the regulation of the tonic activity of inhibitory premotoneurons could
have important consequences for the reflex regulation of airway patency. Inhibitory premotoneurons are located in the NR
(Sumino and Nakamura 1974). Modulation of the ionic currents underlying autorhythmicity in GFP⫹ interneurons could
influence the amount of tonic inhibition that HMs receive and
put their integrative properties under the control of a host of
neuromodulator substances found in the brain stem that can
affect ionic conductances.
In conclusion, our study suggests that GABAergic interneurons in the NR are a source of tonic inhibitory synaptic input
to HMs. The heterogeneity of firing and morphological properties of these cells would suggest that they perform different
tasks. What those tasks are and whether they are active during
different behaviors remain to be established.
ACKNOWLEDGMENTS
We thank J. Numata for expert technical assistance, help with data analysis,
and drawing biocytin-filled cells and L.-S. Burton for drawing biocytin-filled
cells.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-49657 to A. J. Berger and H. van Brederode.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
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