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Phil. Trans. R. Soc. B (2009) 364, 2501–2516
doi:10.1098/rstb.2009.0075
Central and peripheral chemoreceptors evoke
distinct responses in simultaneously
recorded neurons of the raphé-pontomedullary
respiratory network
Sarah C. Nuding, Lauren S. Segers, Roger Shannon, Russell O’Connor,
Kendall F. Morris and Bruce G. Lindsey*
Department of Molecular Pharmacology and Physiology and Neuroscience Program,
School of Biomedical Sciences, College of Medicine, University of South Florida,
12901 Bruce B. Downs Boulevard, Tampa, FL 33612-4799, USA
The brainstem network for generating and modulating the respiratory motor pattern includes
neurons of the medullary ventrolateral respiratory column (VRC), dorsolateral pons (PRG) and
raphé nuclei. Midline raphé neurons are proposed to be elements of a distributed brainstem
system of central chemoreceptors, as well as modulators of central chemoreceptors at other sites,
including the retrotrapezoid nucleus. Stimulation of the raphé system or peripheral chemoreceptors
can induce a long-term facilitation of phrenic nerve activity; central chemoreceptor stimulation does
not. The network mechanisms through which each class of chemoreceptor differentially influences
breathing are poorly understood. Microelectrode arrays were used to monitor sets of spike trains
from 114 PRG, 198 VRC and 166 midline neurons in six decerebrate vagotomized cats; 356
were recorded during sequential stimulation of both receptor classes via brief CO2-saturated
saline injections in vertebral (central) and carotid arteries (peripheral). Seventy neurons responded
to both stimuli. More neurons were responsive only to peripheral challenges than those responsive
only to central chemoreceptor stimulation (PRG, 20 : 4; VRC, 41 : 10; midline, 25 : 13). Of 16 474
pairs of neurons evaluated for short-time scale correlations, similar percentages of reference neurons
in each brain region had correlation features indicative of a specific interaction with at least one
target neuron: PRG (59.6%), VRC (51.0%) and raphé nuclei (45.8%). The results suggest a brainstem network architecture with connectivity that shapes the respiratory motor pattern via overlapping circuits that modulate central and peripheral chemoreceptor-mediated influences on breathing.
Keywords: chemoreceptor reflex; brainstem circuits; breathing
1. INTRODUCTION
The brainstem network for respiratory rhythm and
motor pattern generation includes the medullary
ventrolateral respiratory column (VRC) and the dorsolateral pontine respiratory group (PRG; Marckwald
1887; Lumsden 1923; Stella 1938; Cohen 1979;
St John 1985, 1986, 1998; Smith et al. 1991, 2007;
Dick et al. 1994, 2008; Bianchi et al. 1995; Feldman
et al. 2003; Alheid et al. 2004). A recent study of
functional connectivity within this pontomedullary
network supported model-based hypotheses on circuit
mechanisms for pontine influences on respiratory
phase switching and drive (Rybak et al. 2008; Segers
et al. 2008). Multi-array electrode technology and
spike train analysis have also identified correlational
linkages that support a model of respiratory network
architecture with raphé circuits serving as a parallel
system of ‘intermediate relays’ between the VRC and
PRG (Nuding et al. 2009), as proposed by Bianchi
*Author for correspondence ([email protected]).
One contribution of 17 to a Discussion Meeting Issue ‘Brainstem
neural networks vital for life’.
et al. (1995). These results complement earlier evidence for functional relationships between the VRC
and raphé nuclei, including that for efference copy of
respiratory drive information to midline circuits and
raphé modulation of phase timing and drive, and
support the hypothesis that midline circuits maintain
particular states or levels of neuronal activity that are
subject to adjustment by afferent systems (Lindsey
et al. 1992a,b,c, 1994, 1998; Morris et al. 1996a,b,
2001; Li et al. 1999b; Arata et al. 2000; Aungst et al.
2008).
Medullary raphé neurons respond to perturbations
of both central and peripheral chemoreceptor systems.
State-dependent changes in ventilation in response to
local perturbations of raphé neurons support the
hypothesis that midline neurons are elements of a distributed brainstem system of central chemoreceptors
(Nattie & Li 2009), as well as modulators of central
chemoreceptors at other sites, including the retrotrapezoid nucleus (Mulkey et al. 2004; Dias et al. 2008).
Stimulation of peripheral chemoreceptors also evokes
changes in raphé neuron activity (Morris et al.
1996a,b, 2001), and repeated intermittent stimulation
of peripheral carotid body chemoreceptors or
2501
This journal is q 2009 The Royal Society
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2502
S. C. Nuding et al.
Chemoreception and brainstem circuits
(a)
(c)
∫ phrenic
peripheral
chemoreceptor
stimulation
CN IX
vertebral artery injection
phase
plot
vertebral
artery
central
chemoreceptor
stimulation
carotid bodies
308 s
1
cycle 82
(d)
∫ phrenic
carotid artery injection
95 s
phase
plot
1
cycle 25
(b)
spontaneous
augmented bursts
∫ phrenic
145
blood
pressure
central
central
peripheral
1200 s 60
mm Hg
peripheral
Figure 1. Stimulus protocols and resulting changes in phrenic nerve activity and blood pressure. (a) Carotid chemoreceptors
were stimulated selectively by 30 s injections of a CO2-saturated 0.9 per cent saline solution (range 0.521.0 ml) delivered just
below the sinus via a catheter inserted into the external carotid artery. Central chemoreceptors were stimulated by 30 s injections (trials) of CO2-saturated saline (range 0.821.0 ml) into the left axillary artery at the level of the left vertebral artery.
(b) Integrated phrenic nerve and blood pressure traces during two trials each of central (left) and peripheral (right) chemoreceptor stimulation in the same recording. Note spontaneous augmented phrenic bursts (arrows). (c,d) Detail of integrated
phrenic activity traces during the central (c) and the peripheral (d) chemoreceptor stimulation marked by the boxes in (b).
Phase graphs beneath these traces show the peak amplitude of integrated phrenic activity during cycles occurring before,
during and after the same stimulus period. The dashed and solid horizontal lines indicate the average +2 s.d. of the peak
integrated phrenic amplitude during the interval before stimulation. Neuronal activity was examined for responses if the
accompanying phrenic amplitude increased significantly by this analysis.
medullary raphé neurons can induce a long-term
facilitation of phrenic nerve amplitude and cycling frequency (Millhorn et al. 1980; Morris et al. 1996a,b,
2001; Mitchell et al. 2001). Similar patterns of central
chemoreceptor stimulation do not produce this
respiratory memory (Millhorn 1986).
The network mechanisms through which each class
of chemoreceptor differentially influences breathing
and longer term changes in the respiratory motor pattern are not well understood. Given the results
described above and the roles proposed for medullary
raphé neurons, we addressed the hypothesis that midline brainstem circuits are connected to the PRG and
VRC in a way appropriate for shaping the physiological
responses evoked by the stimulation of both types of
chemoreceptors. Here, we report simultaneous
measurements of the firing rates of neurons in all
three domains during sequential transient stimulation
of central and peripheral chemoreceptors, together
with results from spike train cross-correlation analysis
Phil. Trans. R. Soc. B (2009)
indicative of paucisynaptic associations among them.
A preliminary account of some of the results has
been presented elsewhere (Nuding et al. 2007).
2. METHODS
Data were obtained from six adult cats (2.8 – 5.6 kg) of
either sex as part of a larger study on the brainstem
respiratory network. A detailed description of the
methods has been published elsewhere (Segers et al.
2008). Briefly, animals were initially anaesthetized
with isoflurane (2 – 5%; n ¼ 3) or with an intramuscular ketamine hydrochloride injection (5.5 mg kg21;
n ¼ 3) followed by isoflurane and later decerebrated
using a technique adapted from Kirsten & St John
(1978). The level of anaesthesia was assessed periodically by noxious stimuli (toe pinch); if the withdrawal
reflex occurred or there was an increase in blood
pressure or respiration, the percentage of isoflurane in
the inspired gas was increased until the response was
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Chemoreception and brainstem circuits S. C. Nuding et al.
(b)
max.
spikes s–1
6.4
PRG
CP
E
524
raphé
(i)
16.5
NRM
904
11.7 spikes s–1
(a)
274
0
PSTH
0
–90
14.6 spikes s–1
VRC
41.2
E-Dec-P
358
20.8
I-Dec
362
90 s
2
3
0
90 s
2
0
90 s
peripheral chemoreceptor stimulation
0.008
0.006
0.007
0.005
0.006
0.004
0.003
0.005
0.002
0.004
0.003
0.001
central chemoreceptor stimulation
CUSUM
–217
–90
0.009
0.007
175
mm Hg
75
3
1555 s
peripheral chemoreceptor stimulation
1
90 s
0
PSTH
–90
spikes per tic
1
0
217
(c) central chemoreceptor stimulation
phrenic
blood
pressure
CUSUM
–274
–90
(ii)
14.7
E-other
921
2503
0
56 112 168 224 280 336
0 30 60 90 120 150 180
cycles
cycles
neuron 904
Figure 2. Simultaneously monitored neurons during repeated sequential stimulation of central and peripheral chemoreceptors. (a) Firing rate histograms of five simultaneously monitored neurons, integrated efferent phrenic nerve activity
and arterial blood pressure during repeated sequential stimulation of central (left) and peripheral (right) chemoreceptors.
Arrows to the left of each histogram indicate direction of significant change in firing rate in response to central (C) and peripheral (P) chemoreceptor stimulation: increase ("), decrease (#) or no change (!); see §2 for significance tests. Firing rates
shown on the right refer to the tallest bin (i.e. the maximum rate) in the corresponding histogram. The horizontal black bars
denote intervals when solutions were injected to stimulate central or peripheral chemoreceptors; vertical grey panels highlight
the response periods. The boxed segment of the phrenic and blood pressure traces (shown with an expanded vertical scale
below) indicates data collected during the vertebral injections; arrows denote phrenic-nerve firing rate changes (top trace)
and blood pressure increases (bottom). (b) peri-stimulus time histograms (PSTH) and corresponding cumulative sum histograms (CUSUM) detailing the responses of neuron 904 to (i) central and (ii) peripheral chemoreceptor stimulation. The
PSTH plots the average firing rate of the cell before and following stimulus onset over the three trials shown in (a); stimulus
application began at 0. The CUSUM reflects the data trends seen in the PSTH. (c) Plotted counts of spikes per cycle for three
paired control (white vertical panels) and stimulus periods (grey panels) used to calculate statistics; see §2 for details.
absent. Animals were artificially ventilated through a
tracheal cannula with a respirator. End-tidal CO2,
rectal temperature and arterial blood pressure were
monitored continuously; arterial PO2, PCO2 and pH
were measured periodically. These parameters were
maintained within normal limits. Prior to decerebration, an anaesthetic assessment was performed, animals
were neuromuscularly blocked by pancuronium bromide (initial bolus of 0.1 mg kg21 followed by
0.2 mg kg21 h21, IV) and the brainstem was immediately transected at the midcollicular level. Brain tissue
rostral to the transection was aspirated. Isoflurane was
removed from the inhaled gas circuit after the decerebration was complete. Animals were bilaterally vagotomized to eliminate vagal afferent feedback from
pulmonary stretch receptors and aortic baroreceptors.
At the end of the experiments, cats were killed by an
injection of sodium pentobarbital (28 mg kg21) followed by 5 ml of a saturated solution of KCl in water.
Efferent phrenic nerve activity was monitored
together with signals from three arrays of extracellular
electrodes with individual depth adjustment positioned in the dorsolateral pons (PRG), ventrolateral
medullary respiratory column and medullary midline.
Electrode placement was guided by appropriate stereotaxic coordinates (§3) derived from Berman (1968)
Phil. Trans. R. Soc. B (2009)
and numerous previous studies as described in
Segers et al. (2008) and herein. These signals, together
with systemic arterial blood pressure, tracheal pressure
and end-tidal CO2, were recorded; impulses from
single neurons were converted to arrays of occurrence
times with spike-sorting software.
Two statistical tests were used to evaluate each spike
train for the presence of respiratory-modulated
activity. The first test, a subjects-by-treatments analysis of variance, partitioned each respiratory cycle
(‘subject’ variable) into twenty equal time segments
(‘treatment’ variable; Netick & Orem 1981; Orem &
Netick 1982). A complementary nonparametric
sign test was also used to determine if the probability
of occurrence of an increased firing rate in one half
of the respiratory cycle, over the length of the recording, was greater than chance (Morris et al. 1996a). A
neuron was classified as respiratory modulated if
either test rejected the null hypothesis (p , 0.05);
neurons with no preferred phase of maximum activity
as assessed by both statistical tests were designated
‘non-respiratory modulated’ (NRM). Both standard
and normalized respiratory cycle-triggered histograms
(CTHs) were computed for each neuron and used to
identify the phase (inspiration, I; expiration, E) or
phase transition (IE and EI) in which the cell was
Phil. Trans. R. Soc. B (2009)
10
8
6
Figure 3. (Caption opposite.)
12
4
2
0
R/L
2
4
6
D
8
caudal
rostral
10
6
4
2
0
2
4
6
8
10
12
14
A/P
16
R/
L
8
12
/P
(b)
I-OTH
E-DEC
E-OTH
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
I-DEC
E-DEC-P
NRM
NRM
I-DEC
I-AUG
I-OTH
I-OTH
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
I-DEC
E-DEC-P
NRM
I-DEC
I-DEC
I-DEC
I-DEC
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
E-DEC-P
E-DEC-P
I
I
NRM
NRM
NRM
NRM
I
IE
IE
IE
IE
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
C P
3
C P
I
I
I
I
I
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
2
1
C P
4
C P
I-DEC
I-DEC
I-DEC
I-DEC
I-DEC
I-AUG
I-AUG
E-DEC-P
E-DEC-P
E-DEC-P
E-DEC-P
E-DEC-P
E-DEC-T
E-AUG
I-OTH
E-AUG
E-AUG
E-OTH
E-OTH
E-OTH
EI
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
IE
E
E
E
E
E
E
E
NRM
C P
5
recording
I-DEC
I-DEC
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-OTH
I-OTH
IE
E-DEC-P
E-DEC-P
E-DEC-T
E-DEC-T
E-DEC-T
NRM
NRM
NRM
NRM
I-AUG
I-AUG
I-AUG
IE
IE
E-DEC
E-DEC
E-DEC
E-DEC
E-DEC
E-OTH
E-OTH
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
6
C P
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-OTH
I-OTH
IE
E-DEC-P
E-DEC-T
E-DEC-T
E-DEC-T
E-AUG
EI
EI
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
I-AUG
I-AUG
I-AUG
I-AUG
E-DEC
EI
EI
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
I
IE
EI
EI
NRM
NRM
NRM
NRM
7
C P
I-DEC
I-DEC
I-DEC
I-DEC
I-DEC
I-DEC
I-DEC
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-OTH
I-OTH
E-DEC-P
E-DEC-T
E-DEC-T
E-DEC-T
E-DEC-T
E-DEC-T
E-DEC-T
E-AUG
E-AUG
E-AUG
E-OTH
E-OTH
E-OTH
NRM
NRM
NRM
NRM
NRM
NRM
I-DEC
I-AUG
I-AUG
I-AUG
IE
E-DEC
E-AUG
E-AUG
EI
EI
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
I
I
I
I
I
E
E
EI
EI
EI
NRM
NRM
NRM
NRM
NRM
NRM
NRM
8
I-DEC
I-DEC
I-DEC
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
I-AUG
IE
IE
IE
IE
IE
IE
I-DEC
I-DEC
I-AUG
I-AUG
I-AUG
I-AUG
IE
IE
IE
IE
E-DEC
E-DEC
E-DEC
E-DEC
E-DEC
E-DEC
E-DEC
E-DEC
E-DEC
E-DEC
E-DEC
E-AUG
C P
I
I
I
I
I
I
I
IE
IE
IE
IE
IE
E
E
E
E
EI
EI
EI
NRM
NRM
NRM
E-DEC-P
E-DEC-P
E-DEC-P
E-DEC-T
E-DEC-T
E-DEC-T
E-DEC-T
E-DEC-T
E-DEC-T
E-DEC-T
E-DEC-T
E-DEC-T
E-DEC-T
E-DEC-T
E-AUG
E-OTH
E-OTH
E-OTH
E-OTH
EI
EI
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
NRM
EI
EI
EI
NRM
NRM
NRM
NRM
NRM
NRM
NRM
S. C. Nuding et al.
A
PRG
raphé
2504
VRC
(a)
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Chemoreception and brainstem circuits
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Chemoreception and brainstem circuits S. C. Nuding et al.
2505
Figure 3. (Opposite.) Recording site locations and response profiles of neurons. (a) Dorsal (top) and isometric (bottom) views
of 356 colour-coded spheres indicating the responses and stereotaxic coordinates of neurons tested with sequential stimulation
of central and peripheral chemoreceptors. Neurons monitored at the same coordinates are represented by adjacent vertically
‘stacked’ spheres. The stereotaxic coordinates of the raphé recording sites (n ¼ 125) were within 0.2 mm of the midline and
extended from the obex to 11.6 mm rostral, and from 0.5 to 5.2 mm below the dorsal surface of the medulla. The recording
sites in the PRG (n ¼ 90) ranged from 1.8 mm anterior to 2.0 mm posterior to the caudal border of the inferior colliculus,
2.5–5.8 mm lateral to the midline and 1.3 –3.6 mm below the dorsal surface of the pons. Cells recorded in the VRC (n ¼
141) were located 2.0 mm caudal to 5.2 mm rostral to the obex, 3.0– 4.5 mm lateral to the midline and 2.5 –5.9 mm below
the dorsal surface of the medulla. Units are given in mm. Red circle, neurons responding to central and peripheral chemoreceptor stimulation (n ¼ 70); blue circle, neurons responding to central chemoreceptor stimulation (n ¼ 27); green circle,
neurons responding to peripheral chemoreceptor stimulation (n ¼ 86); grey circle, neurons not responsive to either stimulus
(n ¼ 173); cross, obex. (b) Neuron response profile diagrams from each recording (arranged in columns) reported by region
(raphé, n ¼ 166; PRG, n ¼ 114; VRC, n ¼ 198) and respiratory modulation. Each pair of shaded boxes represents a single
neuron’s responses to central (C) and peripheral (P) sequential chemoreceptor stimulation. Note that in one animal (recordings 1 and 2), only a few responsive neurons were identified, although robust motor responses were evoked in response to the
stimulus protocols. Blue box, increase; red box, decrease; grey box, no change; box with cross, not tested.
more active. In keeping with previous conventions
(e.g. Cohen 1968; Lindsey et al. 1992a; Segers et al.
2008), neurons with peak firing rates in the first half
of the I or E phase were classified as ‘decrementing’
(Dec), whereas those cells with peak firing rates
in the second half of the phase were grouped in the
‘augmenting’ (Aug) subcategory.
Both types of chemoreceptors were selectively
stimulated by close injection of CO2-saturated 0.9
per cent saline solution (pH 5.0–5.2; 0.5–1.0 ml).
Delivery catheters were placed so that blood flow
forced the test solution through the carotid body (peripheral) or through the vertebral artery to the ventral
surface of the medulla (central; figure 1a). Peripheral
chemoreceptors were stimulated by injections delivered
via a catheter inserted into the external carotid artery
and advanced to a point immediately caudal to the carotid sinus (Arita et al. 1988; Li et al. 1999b); sectioning
of the carotid sinus nerve has been shown to eliminate
the phrenic response generated by this established technique for stimulating peripheral chemoreceptors
(Morris et al. 1996a). To stimulate central chemoreceptors, a catheter was inserted into the left axillary artery
and advanced until its tip was in the subclavian artery
proximal to the origin of the left vertebral artery; other
branches off the axillary artery were ligated (Kuwana &
Natsui 1987). Injection of a CO2-saturated saline
solution of similar pH into the vertebral artery
has been shown to affect phrenic nerve activity (e.g.
Hanson et al. 1982; Arita et al. 1988; Seller et al.
1990). Mean systemic arterial blood pressure was
measured before the presentation of each stimulus and
at the point of maximal change following the stimulus.
Most stimulus injections were 30 s in duration; one
recording included 45 s stimulus durations. Neuronal
responses, measured as changes in firing rates, were
initially assessed with conventional peri-stimulus time
(PSTH) and cumulative sum (CUSUM) histograms
(e.g. figure 2b). Changes in activity exceeding CUSUM
confidence bands at +3 s.d. (Davey et al. 1986) were
confirmed by a bootstrap statistical method (e.g.
figure 2c). For each stimulus trial, the first, peak and
maximum contiguous sum of deviations of rate per
respiratory cycle in the response window from the control
mean were used as measures of a response. p-Values were
calculated using ordinary and autoregressive modelbased bootstrap replications (Davison & Hinkley
Phil. Trans. R. Soc. B (2009)
Table 1. Primary responses of single neurons to sequential
central and peripheral chemoreceptor stimulation.
response combinations
location and numbers of
neurons
central
peripheral
PRG
raphé
VRC
"
!
"
#
!
#
"
#
!
Total
"
"
!
#
#
!
#
"
!
6
14
3
2
6
1
0
1
57
90
5
18
8
4
7
5
1
10
67
125
29
26
5
4
15
5
5
3
49
141
1997). The order of the autoregressive model was determined using the finite sample information criterion
(Broersen 2000). The p-value threshold (significance
level) was set by controlling the false discovery rate to a
level of 0.05 (Benjamini & Hochberg 1995).
All pairs of simultaneously recorded spike trains were
evaluated for short-time scale correlations (Perkel et al.
1967); shift-predictor ‘control’ correlograms were
calculated for each pair using 20 cycles at a time with
all possible shifts of these cycles and scaled so that the
mean of the control equalled the mean of the correlogram over a respiratory cycle. Mean shift-control
histograms (+3 s.d.) were superimposed over the
original histogram during analysis. Significant crosscorrelogram features (peaks and troughs) were identified using a ‘detectability index’: the ratio of the maximum amplitude of departure from the background to
the standard deviation of the correlogram noise; features with index values 3 were considered significant
(Aertsen & Gerstein 1985; Melssen & Epping 1987).
Correlation linkage maps for groups of simultaneously
monitored neurons were generated automatically by
database queries using software employing the open
source graph visualization tool GRAPHVIZ. Stereotaxic
coordinates of recording sites were mapped into the
three-dimensional space of a computer-based brainstem atlas derived from The Brain Stem of the Cat: A
Cytoarchitectonic Atlas with Stereotaxic Coordinates, used
E
Phil. Trans. R. Soc. B (2009)
Figure 4. (Caption opposite.)
stimulus
blood
pressure
phrenic
40
I-Aug
I-Aug
41
73
I-Aug
I-Aug
70
65
I-Aug
I-Dec
NT 59
VRC
1216 s
35.9
38.6
27.5
55.7
81.4
42.0
30.1
10.4
32.1
max.
spikes s–1
165
mm Hg
95
E
5s
24.5
32.0
12.3
76.3
71.6
33.3
19.9
0.9
6.3
max.
CTHs spikes
s–1
10 4
10 9
53
59
65
70
73
41
40
8
6
4
2
0
2
4
6
8
10
12
14
16
A
(f)
104
104
–525
–75
65
65
65
65
40
41
73
70
73
65
5
8.6
30.2
525 ms
75 ms
29.6
47.3
22.9
74.0
12 10 8 6 4 2 0 2 4 6 8 10 12 P
L
R
2
max.
(d )
spikes s–1
(b)
–525
104
104
–975
41
65
(e)
–75
59
59
53
(c)
40
41
109
109
70
65
65
4
3
1
525 ms
975 ms
75 ms
17.7
13.7
19.7
19.4
34.3
38.2
45.6
max.
spikes s–1
S. C. Nuding et al.
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53
IE
104
NT 109
P
raphé
central chemoreceptor stimulation
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Figure 4. (Opposite.) Responses to central chemoreceptor stimulation and cross-correlograms selected from one large-scale
multi-array recording. (a) Firing rate histograms for nine simultaneously monitored neurons, integrated phrenic nerve activity
and blood pressure during two trials of central chemoreceptor stimulation; injection durations denoted by bars below analogue
traces. Changes in firing rate in response to stimulation and maximum firing rates for each histogram are shown as in figure 2;
some neurons were not tested (NT) with a particular stimulus. The respiratory modulation of a neuron is included above the
neuron’s ID code and depicted in the normalized CTH to the right of its corresponding firing rate histogram. The CTH of the
neuron (black plot) is shown overlaid upon the CTH of the phrenic nerve (grey plot); the maximum firing rate for each neuronal CTH is shown to the right. For each CTH, 2047 respiratory cycles were averaged and the binwidth was 50 ms; the tic
mark on the x-axis of each CTH indicates the occurrence of the E pulse. Numbers of spikes—neuron 109: 37 390 spikes;
104: 4653; 53: 54 748; 59: 91 313; 65: 134 036; 70: 131 610; 73: 26 550; 41: 37 389; 40: 79 657. (b) Dorsal view of brainstem
showing stereotaxic coordinates of recording sites of neural activity shown in (a). Neuron balls are colour coded according to
their primary response to central chemoreceptor stimulation: red, increase; blue, decrease; grey, no change. (c–f ) Crosscorrelation histograms (CCH) with central and offset features suggestive of functional connectivity among the neurons
depicted in (a). Grey lines in some CCHs represent the mean activity in shifted cycles (§2). Maximum rates are shown
to the right of each CCH. CCH plots were independently offset to facilitate display of multiple plots. Circled numbers
match the particular correlogram features with inferred functional connections shown in figure 6.
with permission of the University of Wisconsin Press, as
described in Segers et al. (2008).
3. RESULTS
Central and peripheral chemoreceptors were selectively
and sequentially stimulated by injection of CO2saturated saline solution into the vertebral and carotid
arteries, respectively (figure 1a). Chemoreceptor reflexes
were identified by a change in the peak integrated phrenic nerve signal amplitude of at least 2 s.d. from the
mean of pre-stimulus values. For central chemoreceptor stimulation trials, the mean time from the start of
the injection to maximum peak phrenic amplitude
was 49.8 + 17.5 s. The corresponding value for
peripheral chemoreceptor trials was 8.4 + 3.3 s.
Phrenic nerve and blood pressure traces during two
trials each of central and peripheral chemoreceptor
stimulation are illustrated in figure 1b. The boxed segments are shown with higher temporal resolution and
different vertical scaling together with corresponding
values of integrated peak activity following arterial
injections (figure 1c,d ). Changes in blood pressure
were associated with some chemosensory stimulus
presentations. The average increase in pressure associated with central chemoreceptor stimulation was 7.0 +
2.6 mm Hg, while that for carotid stimulation was
22.1 + 8.3 mm Hg; both changes were significant
relative to pre-stimulus control values (two-sided
Wilcoxon matched-pairs signed-ranks test, p ¼ 0.02
and p ¼ 0.008, respectively).
Seven of the eight recordings were made with both
vertebral and carotid artery injections. Central chemoreceptors were stimulated first in five recordings.
Peripheral chemoreceptors were stimulated first in
three other recordings to control for possible influences of stimulus order; vertebral injections followed
in two of these recordings. Neurons in each of the primary response categories were found in similar percentages irrespective of stimulus presentation order;
therefore, responses to each type of stimulus were
grouped for analysis.
(a) Identification of responses to
chemoreceptor stimulation
Firing-rate histograms from five neurons recorded simultaneously during sequential stimulation of central
Phil. Trans. R. Soc. B (2009)
and peripheral chemoreceptor are shown together
with integrated efferent phrenic activity and arterial
blood pressure in figure 2a. In this example, central
chemoreceptor stimulation evoked transient declines
in the firing rates of the two raphé neurons (904,
921); no other cell had a significant response when
assessed using measurements from the three trials. In
contrast, all five neurons responded to peripheral chemoreceptor stimulation; both raphé neurons exhibited
increased activity. The PSTH plots the average firing
rate of the cell before and following stimulus onset
over the three trials shown in figure 2a; stimulus application began at 0. Both the PSTH and the CUSUM of
the PSTH (figure 2b) show the average firing rate of
raphé neuron 904 before and after the onset of central
(top) and peripheral (bottom) chemoreceptor stimulation. The responses of raphé neuron 904 to central
and peripheral chemoreceptor stimulation as determined by bootstrap statistics (figure 2c; plotted
counts of spikes per cycle for three paired control
(white vertical panels) and stimulus periods (grey
panels); see §2 for details) were classified as a
‘decrease’ (#) and an ‘increase’ ("), respectively.
Vertebral and carotid artery injections each evoked
biphasic changes in the firing rates of some neurons.
These response patterns were characterized as
increase– decrease (" #) or decrease–increase (# ")
depending on the order of the direction of firing rate
changes relative to pre-stimulus values. Three
examples of biphasic responses (two " # and one
# ") to peripheral chemoreceptor stimulation are
shown in figure 2a. A summary of the responses of
neurons in each brain region to central and peripheral
chemoreceptor stimulation is given in table 1. Owing
to the relative paucity of biphasic responses, evoked
changes in activity were tabulated based on the initial
significant (primary) response direction. We note
that a higher percentage of cells responding to carotid
stimulation exhibited biphasic responses than did
neurons that responded to vertebral stimulation (22/
166, 13% versus 6/99, 6%). Overall, 70 neurons
responded to both stimuli. Of these, 50 (71%) had
firing rate changes in the same direction, with many
more neurons responding with increases (57%)
rather than decreases (14%) in activity. The remaining
20 cells (29%) responded with rate changes in opposite directions, with cells functionally inhibited by
Phil. Trans. R. Soc. B (2009)
VRC
PRG
raphé
"
#
!
"
#
!
2
2
2
0
0
1
2
1
17
"
#
!
"
#
!
"
#
!
1
0
37
!
"
#
!
0
0
0
"
"
#
!
"
#
!
4
2
13
!
0
0
0
"
#
!
0
0
1
#
#
"
#
!
2
0
0
"
peaks
1
2
4
1
1
0
0
3
0
0
0
4
0
0
0
1
0
0
1
0
3
1
1
1
0
1
0
troughs
3
6
11
2
0
3
6
5
6
2
4
7
1
1
0
1
0
0
4
1
17
3
2
4
0
1
0
peaks
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
troughs
VRC
3
3
7
0
2
1
2
2
0
0
2
5
0
0
1
0
0
1
0
0
3
0
0
0
0
0
1
6
1
23
4
1
2
17
3
10
3
1
11
2
0
1
1
1
2
7
5
14
0
0
1
3
1
5
peaks
"
#
!
troughs
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
3
2
14
3
2
0
1
0
2
5
4
9
5
0
3
4
0
28
0
0
0
0
1
11
4
0
14
0
0
1
3
1
7
8
1
3
1
1
3
0
0
2
3
2
2
2
0
1
0
0
2
0
1
2
peaks
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
PRG
raphé
PRG
3
0
6
1
0
1
3
0
1
0
0
3
0
0
0
1
0
1
0
0
5
1
2
0
0
1
0
troughs
8
1
8
9
2
3
7
1
7
7
0
5
2
0
2
2
0
1
11
1
12
1
0
0
5
2
3
peaks
raphé
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
3
2
4
2
1
2
2
1
5
1
0
6
0
0
1
1
0
1
0
0
1
1
0
1
0
0
1
troughs
6
4
21
5
4
3
20
4
10
2
1
9
0
0
2
0
2
6
10
4
11
2
0
0
6
4
6
peaks
VRC
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
"
#
!
4
4
9
5
1
2
7
4
3
2
0
4
2
0
0
3
0
3
3
0
0
1
0
0
1
2
1
troughs
S. C. Nuding et al.
central/peripheral
response of
reference neuron
peripheral
central
2508
location and response of target neuron
Table 2. Summary of offset correlation features for pairs of neurons tested with central and peripheral chemoreceptor stimulation.
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Chemoreception and brainstem circuits S. C. Nuding et al.
vertebral injections and excited by carotid injections
outnumbering cells excited by vertebral injections
and inhibited by carotid injections (14 versus 6).
(b) Response profiles and respiratory-modulated
discharge patterns of simultaneously
recorded neurons in each
brainstem region
Figure 3a shows dorsal and isometric perspectives of
the recording site coordinates of all neurons tested
with sequential stimulation of central and peripheral
chemoreceptors mapped in a standard brainstem
atlas (Berman 1968). All raphé neurons were monitored within 0.2 mm of the midline. Cells recorded
at the same coordinates, either simultaneously with
the same electrode or during different experiments,
are shown as vertically displaced spheres, colour
coded to indicate the stimulus protocol(s) that
evoked responses (central—blue, peripheral—green
or both—red). Neurons without significant responses
are represented by grey spheres.
Response profile diagrams generated for each
recording and arranged by brain region (figure 3b)
show the primary response for each neuron to each
stimulus protocol and the cell’s respiratory-modulated
discharge pattern. The numbers of spike trains monitored simultaneously ranged from 36 to 116 in the
eight recordings from six animals. The use of electrode
arrays with individual depth adjustment allowed testing of many neurons simultaneously and under the
same conditions.
Most of the neurons responsive to both central and
peripheral stimulation were respiratory modulated
(64/70), as were cells responsive only to peripheral
(68/86) or central stimulation (19/27). A majority of
the cells challenged with both stimulus protocols
either did not respond to either stimulus (173/356;
49%) or responded to only one (carotid only: 86/
356, 24%; vertebral only: 27/356, 8%). A subset of
the raphé neurons responded to both peripheral and
central chemoreceptor stimulation; most of these
neurons were respiratory modulated (17/20; 85%).
This percentage was greater than for respiratorymodulated raphé cells responsive only to peripheral
(17/25; 68%) or central chemoreceptor stimulation
(7/13; 54%). Among 19 inspiratory-modulated raphé
neurons, 8 (42%) had increased activity in response
to peripheral chemoreceptor stimulation and 3
(16%) to central chemoreceptor stimulation. Two
were functionally excited by both protocols. Of 37
VRC I-Aug neurons tested, 14 (38%) responded
with increased firing rates to both stimulus protocols.
The activities of 20 (54%) increased during peripheral
chemoreceptor stimulation; 16 (43%) were functionally excited by central chemoreceptor stimulation.
(c) Correlational linkages and
neuronal responses
An aim of spike-train cross-correlation analysis is to
define simple models of neuronal circuits that can
reproduce experimentally observed features (Aertsen
et al. 1989). Using simple model-based inferences,
central peaks and troughs are considered indicative
Phil. Trans. R. Soc. B (2009)
2509
of shared influences of like and opposite sign, respectively; similarly, peaks and troughs offset relative to the
correlogram origin may be interpreted as signs of functional excitation and inhibition (see e.g. Perkel et al.
1967; Moore et al. 1970; Balis et al. 1994; Duffin
2000; for further discussion).
Spike trains from a total of 16 474 pairs of neurons
were evaluated for short-time scale correlations; 522
had primary offset features. For each brain region, the
following numbers (percentages) of reference neurons
had an offset-feature correlation with at least one other
neuron: raphé nuclei (76, 45.8%), PRG (68, 59.6%)
and VRC (101, 51.0%). The left panel in table 2
provides a summary of neuronal responses to central
chemoreceptor stimulation and positive lag offset peaks
and troughs in correlograms calculated using raphé,
PRG or VRC neuron spike trains as reference events,
arranged according to the brain region where the respective target neurons were recorded. The right half of table 2
is similarly organized, but summarizes correlations and
primary responses to peripheral chemoreceptor stimulation. This arrangement is intended to facilitate the
development of model circuits for chemoreceptor reflex
modulation of the respiratory motor pattern. For
example, the top row in table 2 (left panel) indicates
that a total of five correlograms with offset peaks were
identified for pairs composed of raphé reference (trigger)
neurons and PRG or VRC target cells functionally
excited by central chemoreceptor stimulation. Examples
of correlogram features represented in the table are
described and considered below.
Correlation linkage maps generated from spiketrain datasets acquired from neurons monitored at
multiple sites revealed evidence of distributed functional relationships. One group composed of two
raphé neurons with E and IE respiratory-modulated
discharge patterns and seven VRC inspiratory neurons, including two with I-Dec and five with I-Aug discharge patterns, is represented in figure 4a. The raphé
E neuron responded to central chemoreceptor stimulation with a decline in firing rate. The IE neuron
had more variable activity and no specific response
was detected. Each VRC neuron responded to both
central and peripheral (not shown) chemoreceptor
stimulation with increased activity; the I-Dec neuron
designated 59 was not tested for responses to the
latter stimulus because of a loss of signal. Stereotaxic
coordinates of recording sites are shown in figure 4b.
Figure 4c–f shows cross-correlogram features (peaks
and troughs) that departed from background firing
probability, which is represented by the mean shiftpredictor control trace in some plots. The correlograms
in figure 4c were calculated using spikes in I-Dec
neurons 53 and 59 as trigger events and impulses in
two I-Aug neurons (65 and 70) as target events. The
features in the correlograms for pairs 53!65, 59!65
and 59!70 include significant peaks and troughs,
with primary troughs (arrows, circle 1) to the right of
the correlogram origin in all three histograms. Circled
numbers match the particular correlogram features
with inferred functional connections represented in
the ‘ball-and-stick’ models shown in figure 6 (§4).
Central peaks were the primary feature in
correlograms for several pairs of VRC I-Aug neurons;
Phil. Trans. R. Soc. B (2009)
Figure 5. (Caption opposite.)
stimulus
blood
pressure
phrenic
E-Dec-T
117
NRM
114
NRM
136
I-Aug
904
I-Dec
909
I-Plat
912
IE
911
E-Aug
513
I-Plat
503
VRC
raphé
60.1 s
30.0
54.0
14.0
16.0
37.8
33.2
41.3
13.5
10.0
max.
spikes s–1
145
mm Hg
65
E
6s
2.4
36.4
3.9
6.0
17.4
9.2
5.6
0.5
3.7
(f)
–775
513
911
–125
911
909
513
912
–625
114
(h) 114
10
9
7
117
909
775 ms
14.4
13.2
125 ms
12.7
13.5
13
12
–625
909
(g)
–275
912
911
(e)
–525
904
136
(c)
625 ms
117
114
114
503
904
1.3
13.3
11
8
6
1.1
39.6
40.2
625 ms
275 ms
525 ms
3.7
8.7
S. C. Nuding et al.
NT
P
PRG
max.
CTHs spikes s–1
50 3
16 A
14
51 3 12
10
8
91 2
6
911
13 6 4
11 4 2
90 4
11 7 0
90 9
2
4
6
8 P
12 10 8 6 4 2 0 2 4 6 8 10 12
R
L
max.
(d )
spikes
s–1
909 912
(b)
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Chemoreception and brainstem circuits S. C. Nuding et al.
2511
Figure 5. (Opposite.) Responses to peripheral chemoreceptor stimulation and cross-correlograms selected from one large-scale
multi-array recording. (a) Firing rate histograms for nine simultaneously monitored neurons, integrated phrenic nerve activity
and blood pressure during two trials of peripheral chemoreceptor stimulation; labelling conventions are as in figure 4. For each
CTH, 2758 respiratory cycles were averaged and the binwidth was 60 ms. Numbers of spikes—neuron 503: 25 902 spikes; 513:
3526; 911: 49 505; 912: 72 647; 909: 125 495; 904: 51 885; 136: 33 409; 114: 329 666; 117: 9588. (b) Dorsal view of brainstem showing stereotaxic coordinates of recording sites of neural activity shown in (a). Neuron balls are colour coded according
to their primary response to peripheral chemoreceptor stimulation: red, increase; blue, decrease; grey, no change. (c – h) Crosscorrelograms with central and offset features suggestive of functional connectivity among the neurons depicted in (a). Grey lines
in CCHs represent the mean activity +3 s.d. in shuffled cycles. Maximum rates are shown to the right of each CCH. CCH plots
were independently offset to facilitate display of multiple plots. Circled numbers are the same as in figure legend 4.
inferred functional connections
(a)
PRG
(b)
I
E
503
513
9
VRC
VRC
NRM
I-Driver
raphé
IE
I-Dec
911
53
1
I
I-Dec
raphé
912
59
6
3
E
136
I-Dec
10
8
12
909
NRM
109
4
114
I-Aug
I-EI
65
2
1
904
13
I-Aug
70
I-Aug
73
7
shared
drive
11
E-Dec
117
I
Drive r
E-Aug
IE
104
5
I-Aug
I-Dec
41
I-Aug
40
I-Aug
Phr.
MN
diaphragm
Figure 6. Ball-and-stick models of functional connectivity suggested or supported by the results. Simple interpretations of
offset correlogram features and responses to (a) central or (b) peripheral chemoreceptor stimulation for two large-scale
multi-array recordings. Individual neurons are shown as large circles labelled with the cell ID code, respiratory type and
response to chemoreceptor stimulation. The small circles at the ends of the connecting lines indicate the particular synaptic
relationship: excitatory and inhibitory synapses are shown as open and solid circles, respectively. Circled numbers refer to functional connections inferred from correlograms in figures 4 and 5. The circuit shown in the lower half of the VRC in (b) (circles
with no cell ID codes) is a previously published summary of inferred actions among VRC neurons (Lindsey et al. 1998). The
raphé I-EI neuron began its increase in discharge rate shortly before the E-to-I phase transition. Phr. MN, phrenic motor neurons. See text for further discussion. Open circle, offset peak; filled large circle, offset trough; filled small circle, network
junction.
secondary bilateral troughs were also apparent
(figure 4d ). When spikes from two of these I-Aug
neurons served as trigger events in correlograms with
Phil. Trans. R. Soc. B (2009)
the expiratory-modulated raphé neuron 109, two significant peaks with positive lags were identified
(figure 4e, circle 3). Troughs to the left of the peaks
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2512
S. C. Nuding et al.
Chemoreception and brainstem circuits
were also noted (circle 4); both had detectability index
values greater than 3. The offset peaks in each correlogram in figure 4f (arrows) suggest functional connections between one unresponsive raphé IE neuron and
four of the five VRC I-Aug neurons. Simple interpretations of the correlation features together with current
models of the respiratory network suggest the circuit
represented in figure 6a, which is considered further
in §4.
Responses and CTHs from another group of correlated neurons that exhibited various changes in firing
rate during the stimulus periods are shown in
figure 5a. Changes in firing rate during two peripheral
chemoreceptor stimulation trials are illustrated.
Stereotaxic coordinates of recording sites of these
nine neurons are shown in figure 5b. Extended correlational linkages were identified among various neurons
in this group; some are documented by the features
shown in figure 5c – h. For example, the offset peak
with the positive lag in the 136!904 correlogram
(figure 5c) is followed by a trough. The 904!503 correlogram has secondary peaks and troughs in addition
to a primary peak with a positive lag (figure 5c). The
remaining features illustrated in the correlograms
include central peaks (figure 5d ), primary offset
peaks (figure 5e, f,h) and an offset trough (figure 5g).
Simple circuits consistent with this feature set are
shown in figure 6b and considered in §4.
4. DISCUSSION
Overall, the results of this study suggest a brainstem
network architecture with connectivity for the modulation of the respiratory motor pattern by overlapping
circuits that promote and limit central and peripheral
chemoreceptor-mediated influences on breathing.
The response profiles of neurons distributed in the
raphé-pontomedullary respiratory network show that
overlapping populations are differentially influenced
by selective, sequential, transient stimulation of central
and peripheral chemoreceptors. Stimulation of both
classes of receptors resulted in increased VRC, raphé
and PRG inspiratory neuron activity, although particular inspiratory neurons did respond differently to
the two stimuli. Correlations between neuronal spike
trains indicate paucisynaptic interactions among neurons responsive to stimulation of either or both types of
chemoreceptors; a functional projection to at least one
other neuron could be inferred for approximately 50
per cent of the cells in each brain region. Detected correlational linkages suggest multiple feedback loops
between different categories of neurons in the VRC
and cells in the PRG and brainstem midline, supporting recent models of respiratory network architecture
(Smith et al. 2007; Rybak et al. 2008; Nuding et al.
2009).
Sequential observations of single neurons cannot
distinguish between responsive cells with a putative
‘relay’ or excitatory function and neurons with similar
responses that may act to suppress or limit the activity
of their targets. Our use of electrode arrays contributed to experimental efficiency (i.e. more neurons
recorded per experiment) and enabled the detection
of multiple correlations among the simultaneously
Phil. Trans. R. Soc. B (2009)
monitored spike trains. Moreover, for each monitored
group of neurons, changes in activity and the respiratory motor pattern in response to a particular stimulus
were measured under the same history-dependent
conditions, and, therefore, were not confounded by
possible changes in the state of the animal. We also
note that our approach screened neurons for responses
to chemoreceptor perturbations using measurements
of firing rate. Circuit architectures that promote
changes in impulse synchrony with or without changes
in firing rate could also be involved in brainstem sensory processing of chemoreceptor information
(Morris et al. 2001) and would not necessarily be
detected with this approach, as has also been noted
elsewhere in studies on baroreceptors (Arata et al.
2000) and pulmonary stretch receptor reflexes (Dick
et al. 2008) in the control of breathing. This possibility
remains an area for future investigation.
(a) Functional implications
Medullary raphé neurons constitute a major modulatory system in the control of breathing (Holtman
et al. 1986a,b; Lalley 1986b; Millhorn 1986). Both
anatomical and physiological evidence support a role
for the raphé nuclei in respiratory regulation. Numerous anatomical studies in both cat (Lalley 1986a) and
rat (Connelly et al. 1989; Holtman et al. 1990) have
demonstrated projections between raphé nuclei and
other regions of the nervous system involved in
respiratory control. Their putative functions include:
(i) permissive or enabling roles in respiratory rhythmogenesis (Lalley et al. 1997; Lovick 1997; Peña &
Ramirez 2002); (ii) a respiratory memory associated
with increased ventilation in response to repeated
intermittent peripheral chemoreceptor stimulation or
hypoxia (Millhorn et al. 1980; Millhorn 1986;
Morris et al. 1996b, 2001, 2003; Mitchell & Johnson
2003); (iii) baroreceptor modulation of the respiratory
motor pattern (Lindsey et al. 1998); (iv) network
reconfiguration during cough (Shannon et al. 1998,
2000; Baekey et al. 2003); and (v) a central chemoreceptor function (Bernard et al. 1996; Wang et al.
1998, 2002; Nattie 1999; Bradley et al. 2002). More
recently, it has been shown that focal acidification of
the rostral medullary raphé nuclei via microdialysis
of CO2 stimulates breathing (Nattie & Li 2001);
ventilation was unchanged when the same technique
was applied solely to the more caudal raphé obscurus
nucleus (Dias et al. 2008). However, high-CO2
dialysis applied simultaneously to the chemoreceptive
retrotrapezoid nucleus and caudal raphé obscurus
nucleus produced a greater increase in ventilation
than when the retrotrapezoid nucleus alone was
stimulated (Dias et al. 2008), suggesting a modulatory
role for the caudal medullary raphé with respect
to central chemoreception at the retrotrapezoid
nucleus.
Previous results from this laboratory have demonstrated the influence of peripheral chemoreceptors
(Morris et al. 1996a,b, 2001) and baroreceptors
(Lindsey et al. 1998; Arata et al. 2000) and their functional convergence on raphé neurons (Li et al. 1999a).
Other work has shown that raphé neurons change their
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Chemoreception and brainstem circuits S. C. Nuding et al.
discharge patterns when the respiratory network is
reconfigured during cough (Baekey et al. 2001, 2003)
and during the alteration or absence of pulmonary
stretch receptor feedback (Morris et al. 2007).
The present results lend further support to the
hypothesis that raphé neurons are organized into functionally connected groups that regulate breathing. The
ball-and-stick models in figure 6a,b represent simple
circuit configurations suggested by the correlograms
and responses to chemoreceptor stimulation shown
in figures 4 and 5, respectively. Connections in
the models are labelled with circled numbers that
correspond to the supporting correlograms.
The results shown in figure 4c,d and represented in
figure 6a reinforce our previous work within the VRC
(Segers et al. 1987; Li et al. 1999a). The offset troughs
in the correlograms in figure 4c provide evidence for
functional inhibition of I-Aug neurons 65 and 70 by
I-Dec cells (figure 6a, circle 1). The central peaks
in figure 4d are consistent with input(s) shared by
VRC I-Aug neurons 65, 70, 41 and 40; shown in
figure 6a (circle 2), these synchronizing inputs (dashed
lines) are postulated to come from an I-Driver population, described previously as VRC neurons whose
activity increases shortly before phrenic nerve discharge,
peaks in the early part of inspiration and then slowly
decrements before abruptly ceasing at the I-to-E phase
transition (Segers et al. 1987; Morris et al. 1996a). All
four VRC I-Aug cells had an increase in firing rate in
response to central chemoreceptor stimulation.
This study also identified evidence for specific
feedback loops that could influence responses evoked
by chemoreceptor stimulation. The peaks and troughs
to the right and left, respectively, of the midline in
the two correlograms in figure 4e suggest a complex
relationship between a raphé E neuron and two VRC
I-Aug cells. These correlation features are consistent
with facilitatory inputs (circle 3 in figure 6a) from
VRC cells 65 and 41 to raphé neuron 109, which, in
turn, provided inhibitory feedback (circle 4) to the
same VRC cells. This circuit and the decreased discharge rate of neuron 109 in response to vertebral
CO2 injection would serve to promote the response of
the VRC neurons to central chemoreceptor stimulation
via disinhibition of the I-Aug cells. Evidence for excitatory modulation of the same two VRC inspiratory
neurons by a raphé IE neuron that did not respond to
either chemoreceptor challenge (figure 4f; circle 5)
suggests that non-responding inputs can bias the reaction of target neurons to chemoreceptor stimulation.
Bianchi et al. (1995) proposed that midline raphé
circuits serve as ‘intermediate relays’ in PRG –VRC
interactions. Previous results from our laboratory support this hypothesis (Nuding et al. 2009). An example
of an ‘indirect’ pathway between the VRC and PRG
involving intermediary raphé cells is included in the
circuit model in figure 6b (circle 6) and reflects the
correlogram features in figure 5c. Both correlograms
contain an offset peak, suggesting that each reference
neuron had an excitatory effect upon its target cell,
resulting in a VRC!raphé!PRG functional connectivity chain. VRC neuron 136 decreased and raphé cell
904 first increased and then decreased its firing rate in
response to peripheral chemoreceptor stimulation.
Phil. Trans. R. Soc. B (2009)
2513
The delayed portion of the biphasic response of
neuron 904 could be due to the reduction of excitatory
input from cell 136 (disfacilitation).
Our data are consistent with the concept of
efference copy of VRC inspiratory drive serving as a
conduit for the transference of chemoreceptor stimulation effects to raphé neurons (Lindsey et al. 1994).
Results from one group of neurons documented
enhanced inspiratory drive to diverse raphé neurons
that had functional connections with the PRG and
VRC. The central peaks in figure 5d suggest that
raphé neurons 909, 911 and 912 shared an input
source, perhaps from efference copy reflecting changes
in VRC output inspiratory drive (circle 7 in figure 6b);
all three cells responded to peripheral chemoreceptor
stimulation with an increased firing rate.
The correlograms of several neuron pairs in figure 5
provide evidence of convergent and divergent functional connections of raphé neurons. The offset
peaks in figure 5e can be interpreted as a convergent
excitatory effect of raphé neurons 911 and 912 upon
VRC cell 114 (circle 8 in figure 6b). Both of the
raphé cells increased in activity while the discharge
rate of the VRC neuron decreased in response to peripheral chemoreceptor stimulation, suggesting that
the activity decline in the VRC neuron following the
stimulation was limited by the increased rates of the
raphé cells. The offset peak in the upper correlogram
in figure 5f suggests that, in addition to its effect
upon VRC cell 114, midline neuron 911 had a divergent excitatory influence upon PRG cell 513 (circle
9), which, in turn, had an excitatory effect on raphé
cell 909 (figure 5f, circle 10). The intermediate position of cell 513 in this raphé !PRG! raphé loop
could serve to gate the transference of increased
activity from neuron 911 to cell 909 in response to peripheral chemoreceptor stimulation.
The results also support the hypothesis that midline
brainstem neurons are connected to the PRG and
VRC in ways appropriate for shaping the physiological
responses evoked by both types of chemoreceptors.
VRC E-Dec neurons are proposed to play a key role
in the regulation of expiratory duration in current respiratory network models (Rybak et al. 2008); changes
in the firing rate and/or discharge pattern of such a cell
would have a bearing on phase switching and breathing rate. Our data suggest that raphé neurons have a
modulatory effect upon VRC E-Dec cells. Interpretations of the three correlograms in figure 5g,h are
shown in figure 6b as circles 11, 12 and 13 and suggest
that VRC cell 114 had a direct excitatory as well as an
indirect inhibitory, self-limiting effect through raphé
neuron 909 upon VRC E-Dec cell 117. Changes in
the gain of intermediary raphé neuron 909 (by the
loop discussed above, for example) would modulate
the level of functional inhibition imposed upon the
VRC E-Dec cell by neuron 114.
(b) Other relationships to previous studies
Long-term respiratory facilitation can be induced by
repeated brief stimulation of peripheral chemoreceptors or medullary raphé neurons (Millhorn et al.
1980; Morris et al. 1996a,b, 2001; Mitchell et al.
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2514
S. C. Nuding et al.
Chemoreception and brainstem circuits
2001) and is associated with enhanced efficacy of
shared drive, leading to increased synchrony of inspiratory neurons and amplified activity in respiratory
motor neurons. A previous study identified raphé
neurons with transient increases in firing rate at the
onset of carotid chemoreceptor stimulation that subsequently declined as a delayed increase in other
raphé neurons developed (Morris et al. 1996a,b,
2001). These distinct response properties and related
short-time scale correlations led to a ‘ratchet’ model
(Morris et al. 2001, fig. 7c): Transiently responding
neurons contribute to the generation of the facilitated
state during periods of high firing rate, while inhibitory
actions of delayed responders limit the amount of
potentiation induced with each stimulus.
The dissimilar responses of some raphé neurons to
central and peripheral chemoreceptor stimulation and
the connectivity inferred by this study suggest interactions that differentially promote and limit chemoreceptor interactions. We found raphé neurons
stimulated by carotid injection but functionally inhibited by central chemoreceptor activation (e.g.
figure 2a). These distinct responses are consistent
with the proposed role of raphé neurons in the ratchet
hypothesis of long-term facilitation induction and the
inability of central chemoreceptor stimulation to
evoke this respiratory memory (Millhorn 1986). A
related ‘push – pull’ proposal based on raphé cell culture data has been suggested for the regulation of respiration and other pH-sensitive central nervous system
functions by Richerson et al. (2001). Our findings are
also relevant to the observation of Eldridge et al.
(1981) that the magnitude of the response to a chemoreceptor stimulus is inversely related to the level
of pre-existing activity. That earlier work postulated
that this hypoadditive interaction occurs at the level
of the central respiratory controller after convergence
of CO2 inputs but prior to medullary output. Our
new results emphasize the need for further studies to
test the broader hypothesis that midline neuronal
assemblies stabilize and regulate the gain of motor
output in cardiorespiratory control (Lindsey et al.
1992c, 1998; Morris et al. 1996a, 2001).
Experiments were performed under protocols approved by
the University of South Florida’s Institutional Animal Care
and Use Committee and with strict adherence to all
American Association for Accreditation of Laboratory
Animal Care International (AAALAC), National Institutes
of Health and National Research Council guidelines.
We thank Peter Barnhill, Kimberly Ruff, Kathryn Ross,
Andrew Ross, Mackenzie Ott and Carl Strohmenger for
excellent technical assistance. This work was supported by
NIH grants R37NS19814 and RO1NS046062.
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