Group Title: BMC Physiology
Title: Phrenic nerve afferents elicited cord dorsum potential in the cat cervical spinal cord
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Title: Phrenic nerve afferents elicited cord dorsum potential in the cat cervical spinal cord
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Language: English
Creator: Chou,Yang-Ling
Davenport, Paul
Publisher: BMC Physiology
Publication Date: 2005
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Abstract: BACKGROUND:The diaphragm has sensory innervation from mechanoreceptors with myelinated axons entering the spinal cord via the phrenic nerve that project to the thalamus and somatosensory cortex. It was hypothesized that phrenic nerve afferent (PnA) projection to the central nervous system is via the spinal dorsal column pathway.RESULTS:A single N1 peak of the CDP was found in the C4 and C7 spinal segments. Three peaks (N1, N2, and N3) were found in the C5 and C6 segments. No CDP was recorded at C8 dorsal spinal cord surface in cats.CONCLUSION:These results demonstrate PnA activation of neurons in the cervical spinal cord. Three populations of myelinated PnA (Group I, Group II, and Group III) enter the cat's cervical spinal segments that supply the phrenic nerve
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BMC Physiology


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BioMed Central


Research article


Phrenic nerve afferents elicited cord dorsum potential in the cat
cervical spinal cord
Yang-Ling Chout and Paul W Davenport*t


Address: Department of Physiological Sciences, Box 100144, HSC, University of Florida, Gainesville FL 32610, USA
Email: Yang-Ling Chou yangling@ufl.edu; Paul W Davenport* davenportp@mail.vetmed.ufl.edu
* Corresponding author tEqual contributors


Published: 06 May 2005
BMC Physiology 2005, 5:7 doi:10.1 186/1472-6793-5-7


Received: 20 October 2004
Accepted: 06 May 2005


This article is available from: http://www.biomedcentral.com/1472-6793/5/7
2005 Chou and Davenport; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: The diaphragm has sensory innervation from mechanoreceptors with myelinated
axons entering the spinal cord via the phrenic nerve that project to the thalamus and
somatosensory cortex. It was hypothesized that phrenic nerve afferent (PnA) projection to the
central nervous system is via the spinal dorsal column pathway.
Results: A single N I peak of the CDP was found in the C4 and C7 spinal segments. Three peaks
(N I, N2, and N3) were found in the C5 and C6 segments. No CDP was recorded at C8 dorsal
spinal cord surface in cats.
Conclusion: These results demonstrate PnA activation of neurons in the cervical spinal cord.
Three populations of myelinated PnA (Group I, Group II, and Group III) enter the cat's cervical
spinal segments that supply the phrenic nerve


Background
The diaphragm is innervated by the phrenic nerve. The
well-studied motor innervation of the diaphragm by the
phrenic nerve arises from motor neurons in the ventral
horn of the cervical spinal cord (Jammes et al., 1995). The
phrenic nerve motor innervation originates from C4 to C8
spinal segments in the cat. The diaphragm also has affer-
ent innervation carried to the central nervous system by
the phrenic nerve. There are both myelinated and non-
myelinated afferents in the diaphragm. The myelinated
afferents have conduction velocities consistent with
Group la, Ib and II afferents. The diaphragm has relatively
few Group la muscle spindles but a relatively large per-
centage of Group Ib golgi tendon organs (Duron et al.,
1978; Corda et al., 1965a; Goshgarian et al., 1986). Group
II mechanoreceptors have also been reported (Corda et
al., 1965a). Thus, the diaphragm has innervation with
afferents that provide muscle mechanical feedback to the


CNS via the phrenic nerve. However, physiological evi-
dence of phrenic afferent activation of the spinal cord dor-
sal horn is lacking.

The role of phrenic afferents in the regulation of dia-
phragm function has been studied with early observations
suggesting that phrenic afferents in the diaphragm are not
involved in controlling the respiratory muscle activity
(Sant'Ambrogio et al., 1962; Corda et al., 1965b; Kohr-
man et al., 1947; Landau et al., 1962). However, electrical
and mechanical stimulation of phrenic afferents are
reported to activate thalamic neurons (Zhang et al., 2003)
and elicit neural activity in the cat somatosensory cortex
(Davenport et al., 1985). In addition, Zechman et al
(1985) reported a correlation of transdiaphragmatic pres-
sure (Pdi) and the perception of inspiratory loads. Knafelc
and Davenport reported that increased Pdi correlated with
the amplitude of the respiratory related evoked potential


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recorded from the human somatosensory cortex. Thus,
there appears to be a projection of diaphragmatic afferents
to the CNS and mechanical changes in the diaphragm cor-
relate with somatosensory activation of the cerebral cor-
tex. However, PnA activation of spinal sensory pathway(s)
remains unknown.

Anatomical studies have found that phrenic nerve affer-
ents (of unknown type) terminate in the dorsal horn lam-
ina I-IV of C4 and C5 spinal cord in rat (Goshgarian et al.,
1986; Malakhova et al., 2001). In a brief report, Larnicol
et al (1984) reported immunoflourescent evidence of dor-
sal root entry of phrenic nerve afferents in the dorsal horn
of the cervical spinal cord. Gill et al (1963) also demon-
strated phrenic motorneuron activities were elicited by
segmental phrenic nerve afferents. Corda et al (1965)
demonstrated that the cervical spinal dorsal rootlets con-
tain diaphragmatic mechanoreceptors, including muscle
spindles and Golgi tendon organs. Electrophysiological
studies have confirmed that Group I and Group II phrenic
nerve afferents project to lateral reticular nucleus (Macron
et al., 1985), external cuneate nucleus (Marlot et al.,
1985), and both ventral and dorsal respiratory-related
areas of brainstem (Macron et al., 1986; Speck et al.,
1987). Moreover, in anesthetized cats, short-latency
responses have been recorded in the thalamus (Zhang et
al., 2003) and somatosensory cortex (Davenport et al.,
1985) after electrical stimulation of the phrenic nerve
afferents and mechanical probing of the diaphragm. If
diaphragmatic proprioceptors project to higher somato-
sensory brain centers via pathways similar to Group la
and Group Ib receptors (Landford and Schmidt, 1983),
then phrenic afferents should enter the spinal cord ipsilat-
erally through the dorsal roots of the cervical spinal seg-
ments, terminate on dorsal horn neurons, dorsal horn
neurons should then project centrally via the dorsal col-
umns to the brainstem and then project to the somatosen-
sory cortex and other supraspinal structures. If this is the
phrenic afferent pathway to the somatosensory cortex,
then electrical stimulation of the phrenic nerve will stim-
ulate phrenic afferents and activate dorsal horn neurons.
However, the activation of the cervical dorsal horn by
stimulation of phrenic afferents has not been reported.

One method to investigate dorsal horn neuronal activa-
tion by afferent stimulation is recording the cord dorsum
potential (CDP) (Yates et al., 1985). Simultaneous stimu-
lation of peripheral afferents activates groups of dorsal
horn neurons. The activation of a group of neurons pro-
duces a dipole referenced to the surface of the spinal cord.
This dipole is the result of a change in polarity of the acti-
vated neurons creating a current flow with the cord sur-
face. Thus, neurons in the dorsal horn of the spinal cord
generate a field potential when an afferent volley arrives.
These negative voltage evoked potentials generated from


the dorsal horn neurons of the spinal cord by stimulation
of peripheral nerves have been extensively studied for
limb afferents (Yates et al., 1982; Manjarrez et al., 2002).
The CDP was first discovered and described by Grasser
and Graham (1933) as they recorded complex evoked
potentials from the surface of the spinal cord. This poten-
tial appeared to be largest in the dorsal horn grey matter
and is generated by a synchronous activation of a popula-
tion of dorsal horn neurons that respond to stimulation
of low-threshold cutaneous afferents.

Stimulation of nerve afferents at an intensity that activates
only Group I muscle afferents has been shown to evoke a
dorsal cord field potential consisting of a triphasic spike,
a short duration negative wave, and a positive wave. Acti-
vation of Group II muscle afferent fibers resulted in a sec-
ond short duration negative component of the CDP
(Bernhard, 1953; Coombs et al., 1956). When a nerve is
stimulated sufficiently distal to the spinal cord, the depo-
larization of the dorsal horn neurons occurs sequentially
as a function of the arrival of afferents with different con-
duction velocities. Thus, it has been shown that the
triphasic spike of the CDP occurs because of a separation
of activation due to the arrival of Group la, Group Ib and
Group II afferents. The different peak latencies allow for
the determination of different populations of activated
afferents.

We reasoned that, if the phrenic nerve contains Group la,
Group Ib and Group II afferents, then stimulating the
phrenic nerve as far distal from the spinal cord as possible
(near the diaphragm) would elicit multiple CDP peaks. In
addition, we hypothesized that if phrenic nerve afferents
enter a cervical segment of the spinal cord, then stimulat-
ing PnA will elicit a CDP in that segment. However, the
CDP for phrenic nerve afferents has never been reported.
Therefore, recording the phrenic afferent CDP was
hypothesized to provide evidence of segmental dorsal
horn activation by phrenic afferents. This study recorded
the CDP from C4 to C7 elicited by stimulating PnA to
determine the cervical spinal segmental distribution of
PnA elicited CPD, to characterize CDP latencies and infer
the populations of PnA eliciting the CDP in cats.

Results
In all cats, electrical stimulation of the phrenic nerve elic-
ited CDP's recorded at the dorsal surface of C4 to C7 cer-
vical spinal segments, and at rostral, middle and caudal
locations within each spinal segment. No CDP was
observed in the C8 spinal segment. A primary CDP elic-
ited by stimulation of PnA was observed and recorded in
dorsal surface of C4 to C7 cervical spinal segments (Fig.
1). The N1 CDP was recorded in the C4 to C7 spinal seg-
ments; whereas three CDP peaks (NI, N2 and N3) were
identified only in the C5 and C6 spinal segments (Fig. 2).


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-700 -


-600 -


-500 -

-400

-300

-200 -

-100 -


0 -


Stimulus artifact


Time (ms)



Figure I
The cord dorsum potential in response to phrenic nerve afferent stimulation in the C5 segment. The trace was the average of
32 stimulus epochs for the middle C5 segment from one animal. The enlarged area expands the large initial peak of the CDP to
better illustrate the N I and N2 peaks. The first negative wave indicates the electrical stimulus used as the zero time point for
peak latency analysis. The stimulus artifact is followed by three negative peaks N I, N2, and N3. The amplitudes of the negative
peaks were measured from the voltage difference between the peak voltage and the averaged voltage of the 5 ms period
before the stimulus onset.


The distributions of the onset latencies and peak ampli-
tudes of individual cervical spinal cord segments are sum-
marized in (Fig. 3) and (Fig. 4), respectively. The averaged
N1 peak latency was 1.7 0.1 ms for all cervical spinal
segments. The averaged N2 peak latency was 2.3 + 0.1 ms
and the averaged N3 peak latency was 4.6 0.3 ms in the
C5 and C6 segments. The averaged conduction velocity
was 94.1 + 8.6 m/sec for the N1 peak, 70.6 7.5 m/sec for
the N2 peak and 35.0 + 3.8 m/sec for the N3 peak. There
was a significant difference between the latencies for N1,
N2 and N3 peaks (P < 0.05). The average N1 peak ampli-


tude was 22.8 6.0 tV in the C4 segment and significantly
less than the N1 peak amplitude for C5, C6 and C7 spinal
segments (p < 0.05). The average N1 peak amplitude was
141.3 + 12.1 [V in the C5 segment and 146.0 + 27.2 pV in
the C6 segment and not significantly different. The aver-
age N1 amplitude was 54.6 30.1 pV in the C7 segment
and significantly less than C5 and C6. N2 and N3 peaks
were only observed in the C5 and C6 spinal segments and
the averaged N2 peak amplitude was 79.2 10.3 tV in the
C5 segment and 82.1 6.5 pV in the C6 segment. The
averaged N3 peak amplitude was 72.0 4.4 XtV in the C5


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N2

/


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-250-
-200-

-150-

-100-

-50-
0-

50-

100-


-250-
-200-
a -150-
-100-
,. -50-
E 0-
0.
S50-

100-






-250-

5" -200-
0 -150-

-100-
a.
E
S-50
0

50-

100-


N1



N2 N3


9 -200-

-150



S-50

0-


I I I I I I I I
0 2 4 6 8 10 12 14

Time (ms)


C5
N1



N2
/ N3





stimulus


0 2 4 6
Time (ms)


I I 1 1
8 10 12 14


Figure 2
Representative illustration of phrenic nerve afferents stimulation related cord dorsum potential recorded at C4, C5, C6, and
C7 spinal segments in one animal. The traces were the average of 32 stimulus epochs for each spinal segment. The N I peak
was recorded in all cervical spinal segments (C4 to C7); whereas N2 and N3 CDP peaks were identified only in C5 and C6 spi-
nal segments. The amplitudes of the negative peaks were measured from the voltage difference between the peak voltage and
the averaged voltage of the 5 ms period before the stimulus onset.


segment and 69.0 11.3 pV in the C6 segment. The
amplitudes of the CDP peaks were significantly different
between the N1, N2 and N3 peaks (P < 0.05).

Discussion
Neural activity was elicited by stimulation of PnA in C4 to
C7 dorsal cervical spinal cord segments in the present
study. The electrical stimulation of PnA served the impor-
tant purpose of demonstrating the existence and locations
of PnA- elicited CDP in C4 to C7 segments of the cat cer-
vical spinal cord. This stimulation of PnA was initially
observed by one or three negative peaks depending on dif-


ferent segments recorded. The presence of the three nega-
tive peaks in C5 and C6 spinal segments appears to be due
primarily to the spinal input from different groups of PnA.
The presence of only the N1 peak in C4 and C7 demon-
strates a different number of afferent populations between
the cervical spinal segments that contribute to the cat
phrenic nerve.

Central projections of PnA
CDP recordings are evidence of the activation of neurons
in the dorsal horn of the cervical spinal cord by PnA. Neu-
ral activity in the dorsal surface of the cervical spinal cord



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0 2 4 6 8 10 12 14

Time (ms)


stimulus


0 2 4 6 8 10 12 14
Time (ms)


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CDP Peak Latency


5.5

5

4.5

C4

33.5


w2.5

2

1.5

1

0.5


E N1
1N2
*N3


rostral middle caudal


C4 C4 C5 C5 C5 C6 C6 C6 C7 C7 C7

spinal segment

Figure 3
Histogram of the CDP peak latencies in different regions of the cervical spinal segments (C4 to C7) after phrenic nerve affer-
ents stimulation in cats. The intensity of the stimulation was 500 pA. N I is the first peak latency. N2 and N3 are the second
and third peak latencies found in C5 and C6 only. indicates a significant differences between N I and N2 peaks. t indicates a
significant difference between N2 and N3 peaks in the C5 and C6 segmental locations. # indicates a significant difference
between NI and N3 peaks in the C5 and C6 segmental locations.


elicited by PnA found in this study is consistent with the
previous studies of limb muscle afferents (Yates et al.,
1985). These results are consistent with PnA entering the
cervical spinal cord, projecting to the dorsal horn, which
then relays PnA to the brainstem, and projects phrenic
afferent information to the somatosensory cortex via a
thalamocortical pathway (Zifko et al., 1995; Davenport et
al., 1985; Zhang et al., 2003). The function of this putative
pathway could be related to the proprioceptive control of
respiratory muscles and respiratory movement control
originating in the motor cortex (Frazier et al., 1991). Dav-
enport et al (1985) proposed that the PnA projection to


the postcruciate region of the cerebral cortex may play a
role in higher brain center control of the respiratory
pump. The sensory projection sites of the PnA were local-
ized in area 3a and 3b of the sensorimotor cortex in cats.
However, the PnA sensory sites in the cortex are not co-
localized with motor sites. This means that the cortical
regions receiving the sensory information from PnA are
separated from the cortical regions of motor output to the
phrenic spinal motor neurons. Recordings of evoked
potentials using phrenic nerve stimulation provide a
unique method for studying the potential pathways for
cortical integration of respiratory afferent information


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rostral caudal rostral middle caudal rostral middle caudal


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CDP Peak Amplitude


250



200


~=50


rostral caudal rostral middle caudal rostral middle caudal rostral middle caudal

C4 C4 C5 C5 C5 C6 C6 C6 C7 C7 C7

spinal segment

Figure 4
Histogram of the CDP peak amplitudes in the cervical spinal segments (C4 to C7) after phrenic nerve afferent stimulation in
cats. N I is the first peak amplitude observed in the C4 to C7 spinal segments; N2 and N3 are the second and third peak ampli-
tudes only observed in the C5 and C6 spinal segments. indicates a significant differences between N I and N2 peaks in the C5
and C6 segmental locations. # indicates a significant difference between NI and N3 peaks in the C5 and C6 segmental loca-
tions. 0o indicates significant differences between the spinal segments for NI peak.


and the projections of PnA to the central nervous system
(Straus et al., 1997). Although the PnA projection path-
ways to the cerebral cortex remain unknown, the present
study supports a dorsal column mediated pathway that
likely involves a multisynaptic thalamic relayed projec-
tion (Zhang et al., 2003). It has been shown in the previ-
ous studies (Frazier et al., 1991) that C-fiber afferents have
much higher threshold and longer latency for PnA stimu-
lation than group la, Ib and group II afferents. Conduc-
tion velocities for C-fibers are less than 1 m/s. With the
length of the phrenic nerve for these cats, the latency for a
C-fiber elicited peak of the CDP would be approximately


30 ms, longer than the 25 ms sampling time used for
recording the post-stimulus epochs in this study. In addi-
tion, the use of 0.1 ms stimulus pulse width does not elicit
c-fiber action potentials. Therefore, it is unlikely that C
fiber afferents contributed to the CDP in this experiment.

PnA elicited CDP
Results of this study provide evidence that a spinal CDP
was elicited by phrenic nerve afferents input to the dorsal
horn of the cat cervical spinal cord. This result is consist-
ent with the report from Cuddon et al (1999) that the
CDP is a measurement of spinal segmental interneurons


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and dorsal horn cell function. The rationale behind the
CDP measurement as an assessment of sensory nerve
afferent and dorsal horn neuronal functions is the phases
of the CDP reflect the different population of afferent fib-
ers activating dorsal horn neurons (Yates et al., 1982;
Yates et al., 1985). The large negative peak (NI) represents
the interneuronal depolarization of spinal dorsal horn
neurons elicited by large myelinated sensory afferents
(Group la & Ib). The activation of a N1 peak in C4 to C7
spinal segments suggests a broadly distributed input to
the spinal cord from phrenic group I afferents (Corda, et
al., 1965). A N1 peak was not recorded in the C8 spinal
segment. There is a second negative peak (N2) that repre-
sents the depolarization of spinal dorsal horn neurons by
slower conducting myelinated sensory afferents (Group
II). A third negative peak (N3) most likely represents the
depolarization of spinal dorsal horn neurons elicited by
small myelinated sensory afferents (Group III). The pres-
ence of N2 and N3 in the C5 and C6 spinal segments sug-
gests a preferential input of group II and group III
afferents into these specific spinal segments.

Limb group la, Ib and II have been shown to project to
somatosensory cortex of cat via a dorsal column pathway
(Jones et al., 1980). Corda et al (1965) reported group la,
Ib and II afferents in the phrenic nerve. It is very likely that
PnA have a projection similar to limb proprioceptors. This
conjecture is supported in the present study by the CDP
elicited in the dorsal surface of cervical spinal cord by
stimulation of PnA which is consistent with a dorsal col-
umn central neural projection pathway. It is therefore
concluded that group la, group Ib, group II and possibly
group III afferents elicit a CDP that is consistent with an
ascending phrenic sensory pathway via the dorsal column
and the dorsolateral funiculus (spinocervical tract) of the
cervical spinal cord.

Somatosensory PnA pathway
Cortical projection of PnA has been shown in both corti-
cal evoked potentials (Zhang et al., 2003; Davenport et al.,
1985) and retrograde fluorescent (Yates et al., 1987) stud-
ies. Activation of the somatosensory cortex in cats after
electrical stimulation of the contralateral phrenic nerve
(Davenport et al., 1986) and intercostals muscles (Daven-
port et al., 1993) has been reported by this laboratory.
One role of the somatosensory projections from phrenic
nerve afferents may be to provide the sensory feedback to
the cerebral cortex of respiratory pump function. The dia-
phragm, the intercostal muscles and accessory muscles of
respiration provide the inspiratory pumping force for ven-
tilation. Stimulation of PnA has been shown in humans to
elicit somatosensory cortical evoked potentials (Zifko et
al., 1996). Inspiratory occlusion produces a maximal load
on the pumping action of the respiratory muscle and has
been reported to elicit somatosensory respiratory related


evoked potential (RREP) in humans (Davenport et al.,
1986; Davenport et al., 2000) and lambs (Davenport et
al., 2001). Knafelc and Davenport reported a correlation
between RREP amplitude and the magnitude of the
increase in Pdi when graded inspiratory resistive loads
were applied in humans. These reports suggest that
mechanical loading of the respiratory muscles, including
the diaphragm, can elicit somatosensory cortical neural
activity. Although the afferents mediating these evoked
potentials are unknown, it is likely that respiratory muscle
afferents are one population of receptors that mediate
these responses. The results presented in the present study
are therefore consistent with the hypothesized role of PnA
in the somatosensation of inspiratory loads. Thus, neu-
rons in dorsal spinal cord activated by stimulation of PnA
may be related to respiratory muscle proprioception, sim-
ilar to what has been found in other muscle systems.

Conclusion
In summary, the present study recorded a spinal CDP elic-
ited by the activation of phrenic nerve afferents. The PnA
project to dorsal horn neurons in the cervical spinal cord
from C4 to C7 indicating these segments can function as
a relay for the conduction of proprioceptive information
from the diaphragm to the higher brain centers in cats.
The first peak, N1, conduction velocity is consistent with
large myelinated afferent activation, group la and group
lb. These PnA enter all the spinal segments that contribute
to the phrenic nerve in cats. The second peak conduction
velocity is consistent with myelinated afferents, group Ib
and large group II. The third peak conduction velocity is
consistent with myelinated afferents, group III. The sec-
ond and third peaks of the CDP were observed only in the
primary spinal origin (C5 and C6 segments) of the cat
phrenic nerve. The CDP potentials described in this study
reflect the first relay by the dorsal spinal cord of the pro-
jection of myelinated PnA to the higher brain centers in
cats. Therefore, the results of this study support the
hypothesis that PnA activation of neurons in the dorsal
cervical spinal cord may be involved in the central projec-
tion of respiratory muscle afferent information.

Methods
General preparation
Experiments were carried out in adult cats (2.5-3.0 Kg).
The University of Florida, Institutional Animal Use and
Care Committee reviewed and approved this study. CDP
recordings were made from cervical spinal segments C4,
C5, C6, C7, and C8 in anesthetized, paralyzed, and
artificially ventilated animals. Adult cats (n = 7) of either
sex were anesthetized with inhalation of halothane-oxy-
gen. The femoral artery and vein were catheterized. Gas
anesthesia was then replaced with a-chloralose by slow
i.v. infusion (25 mg/ml). The animals were tracheot-
omized, vagotomized and placed prone with the head


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and spine fixed into a stereotaxic apparatus. The body
temperature was monitored with a rectal probe and
maintained at 38 1 C with the periodic use of a heat-
ing pad. Arterial blood pressure, expired CO2 and tracheal
pressure were continuously monitored on a polygraph.
The animals were connected to a mechanical ventilator
and paralyzed (gallamine triethiodide). If fluctuation in
blood pressure and heart rate were observed, all experi-
mental procedures were suspended and supplemental
anesthesia was administered (0.1 mg/kg iv per dose) until
a surgical plane of anesthesia was reestablished. The lungs
were periodically inflated to prevent atelectasis. Arterial
blood gases and pH were measured and maintained
within the normal range. The animals received a continu-
ous infusion of lactated Ringers.

Protocol
The skin and muscles overlying the right lower ribs were
incised. The 7t intercostal space was identified and
opened by cutting the intercostal muscles. The intercostal
space was opened with retractors. The right phrenic nerve
caudal to the heart was identified, isolated and dissected
free of the surrounding tissue. The phrenic nerve, about 1
cm cranial to its entry into the diaphragm, was placed
across bipolar platinum stimulating electrodes. The cath-
ode electrode was about 5-7 mm proximal to the anode
electrode. Supramaximal single pulse stimuli (250-500
pA) were delivered at a rate of 0.6 Hz with stimulus dura-
tion at 0.1 milliseconds. The pulse simultaneously trig-
gered the signal average for collecting the 25 msec. post-
stimulus spinal activity sample. The entire surgical field
was covered with saline soaked gauze.

Spinal laminectomies were performed to expose the dor-
sal spinal cord from C2-T2. The dura was reflected. The
exposed spinal surface was flooded with warm mineral
oil. A silver-silver chloride ball electrode was lowered to
the pial surface. This electrode was used to record the
CDP. The electrode was connected to a high impedance
probe, which was connected to an amplifier. The electrical
signal was band pass filtered at 3 Hz 3.0 kHz and ampli-
fied. The amplifier output was led into a signal average
(Model 1401, Cambridge Electronic Design, Ltd) At least
64 post-stimulus epochs were sampled at 10 kHz and
recorded to provide a minimum of 32 evoked spinal
epochs averaged by the computer system to obtain the
CDP (Signal2, Model 1401 Cambridge Electronics Ltd.).
The recording electrode was placed on the surface of the
ipsilateral dorsal spinal cord medial to the dorsal root
entry zone of the spinal segments. Each spinal segment
was subdivided into 3 recording regions: rostral, middle
and caudal based on counting the dorsal roots. The elec-
trode was systematically moved over the spinal surface at
each recording site between C4 and C8. The ground elec-
trode was placed over a bony prominence. Phrenic nerve


stimulation was performed at each point and the CDP
recorded.

Data analysis
A minimum of 32 evoked epochs were averaged by a com-
puter system (Signal2 Cambridge Electronics Ltd.) to
obtain the CDP. The averaged CDP's were analyzed for
peak presence and polarity (Signal-2, Cambridge Elec-
tronics Ltd). The peaks of the CDP were negative voltage
changes that were initially identified in C5 (Fig. 1) and C6
recordings. The peaks were identified and then labeled
based on latency ranges from the known conduction
velocities of phrenic nerve afferents (Corda et al., 1965)
and CDP peak analysis reported for limb afferents (Yates
et al., 1982). The corresponding onset and peak latencies
and amplitudes were determined. The 0-peak amplitudes
were measured from the voltage difference between the
peak voltage and the averaged voltage of the 5 msec
period before the stimulus onset. The initial post-stimulus
baseline was corrected for any DC offset. The means and
standard errors for onset and peak latencies and ampli-
tudes were then calculated. The onset latency of each CDP
was measured from the onset of the stimulus to the start
of first peak of the CDP. The latency of the first negative
peak (N1) of each CDP was measured as the time from the
stimulus to the peak. When present, the latency of the sec-
ond negative peak (N2) and the third negative peak (N3)
were measured in the same manner. The length of phrenic
nerve was measured from the stimulating electrodes to the
dorsal root entry zone to calculate the conduction velocity
of the phrenic nerve afferents. Each component of CDP
was averaged and statistical analysis was applied. The
mean standard deviation (SD) was calculated for all
CDP peak latencies. One-way repeated measure ANOVA
was used to compare between the N1 peak, N2 peak, and
N3 peak latencies and amplitudes from each recording
site. The criterion for significance was p < 0.05.

Abbreviations
PnA Phrenic nerve afferents

CDP cord dorsum potential

C4, C5, C6, C7, C8 Cervical spinal segments

CNS Central nervous system

Pdi Transdiaphragmatic pressure

CO2 Carbon dioxide

Authors' contributions
YL performed the data analysis and drafted the manu-
script. PD carried out the study and coordinated its design.
Both authors read and approved the final manuscript.


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