Anatomical and functional connections between the locus coeruleus and the nucleus tractus solitarius in neonatal rats

Abstract

This study was designed to investigate brain connections among chemosensitive areas in newborn rats. Rhodamine beads were injected unilaterally into the locus coeruleus (LC) or into the caudal part of the nucleus tractus solitarius (cNTS) in Sprague-Dawley rat pups (P7-P10). Rhodamine-labeled neurons were patched in brainstem slices to study their electrophysiological responses to hypercapnia and to determine if chemosensitive neurons are communicating between LC and cNTS regions. After 7-10 days, retrograde labeling was observed in numerous areas of the brainstem, including many chemosensitive regions, such as the contralateral LC, cNTS and medullary raphe. Whole-cell patch clamp was done in cNTS. In 4 of 5 retrogradely-labeled cNTS neurons that projected to the LC, firing rate increased in response to hypercapnic acidosis (15% CO2), even in synaptic blockade medium (high Mg(2+)/low Ca(2+)). In contrast, 2 of 3 retrogradely-labeled LC neurons that projected to cNTS had reduced firing rate in response to hypercapnic acidosis, both in the presence and absence of synaptic blockade medium. Extensive anatomical connections among chemosensitive brainstem regions in newborn rats were found and at least for the LC and cNTS, the connections involve some CO2-sensitive neurons. Such anatomical and functional coupling suggests a complex central respiratory control network, such as seen in adult rats, is already largely present in neonatal rats by at least day P7-P10. Since the NTS and the LC play a major role in memory consolidation, our results may also contribute to the understanding of the development of memory consolidation.

Full text

ANATOMICAL AND FUNCTIONAL CONNECTIONS BETWEEN THE
LOCUS COERULEUS AND THE NUCLEUS TRACTUS SOLITARIUS
IN NEONATAL RATS
L. T. LOPES,
a
L. G. A. PATRONE,
a
K.-Y. LI,
b
A. N. IMBER,
b
C. D. GRAHAM,
b
L. H. GARGAGLIONI
a
AND R. W. PUTNAM
b
*
a
Dept of Animal Morphology and Physiology. Sa
˜o Paulo
State University, FCAV, Jaboticabal, SP, Brazil
b
Dept of Neuroscience, Cell Biology and Physiology, Wright State
University, Dayton, OH, USA
Abstract—This study was designed to investigate brain
connections among chemosensitive areas in newborn rats.
Rhodamine beads were injected unilaterally into the locus
coeruleus (LC) or into the caudal part of the nucleus trac-
tus solitarius (cNTS) in Sprague–Dawley rat pups
(P7–P10). Rhodamine-labeled neurons were patched in
brainstem slices to study their electrophysiological
responses to hypercapnia and to determine if chemosensi-
tive neurons are communicating between LC and cNTS
regions. After 7–10 days, retrograde labeling was observed
in numerous areas of the brainstem, including many
chemosensitive regions, such as the contralateral LC,
cNTS and medullary raphe. Whole-cell patch clamp was
done in cNTS. In 4 of 5 retrogradely labeled cNTS neurons
that projected to the LC, firing rate increased in response
to hypercapnic acidosis (15% CO
2
), even in synaptic
blockade medium (SNB) (high Mg
2+
/low Ca
2+
). In contrast,
2 of 3 retrogradely labeled LC neurons that projected to
cNTS had reduced firing rate in response to hypercapnic
acidosis, both in the presence and absence of SNB.
Extensive anatomical connections among chemosensitive
brainstem regions in newborn rats were found and at
least for the LC and cNTS, the connections involve some
CO
2
-sensitive neurons. Such anatomical and functional
coupling suggests a complex central respiratory control
network, such as seen in adult rats, is already largely
present in neonatal rats by at least day P7–P10. Since
the NTS and the LC play a major role in memory
consolidation, our results may also contribute to the
understanding of the development of memory
consolidation. Ó2016 IBRO. Published by Elsevier Ltd.
All rights reserved.
Key words: chemosensitivity, development, electrophysiol-
ogy, hypercapnia, retrograde labeling, memory consolidation.
INTRODUCTION
The respiratory system is not mature at birth, but
undergoes significant postnatal development (Mortola,
2001). A critical period exists around postnatal day (P)
12–13, when an imbalance between heightened inhibition
and suppressed excitation is evident both neurochemi-
cally as well as electrophysiologically (Wong-Riley et al.,
2013). Likewise, the ventilatory response to hypercapnia
changes as a function of neonatal development
(Putnam et al., 2005). The CO
2
response is vigorous in
the first postnatal days (P1–P5), but then decreases
and reaches its lowest point at P8. Subsequently, the
response rises until P21, when it seems to achieve the
adult level (Stunden et al., 2001; Putnam et al., 2005;
Greer, 2012).
There is growing evidence suggesting that respiratory
central chemoreception is a distributed property including
multiple brainstem regions and neuronal types that may
change over development (Coates et al., 1993; Nattie,
1999; Nattie and Li, 2002, 2009; Putnam et al., 2004;
Richerson, 2004; Biancardi et al., 2008; Gargaglioni
et al., 2010; Hodges and Richerson, 2010; Putnam,
2010). Among these chemosensitive sites are the locus
coeruleus (LC) (Elam et al., 1981; Pineda and
Aghajanian, 1997; Filosa et al., 2002; Gargaglioni et al.,
2010) and the caudal region of the nucleus tractus solitar-
ius (cNTS) (Dean et al., 1989, 1990; Nattie and Li, 2002;
Conrad et al., 2009; Dean and Putnam, 2010). A high
percentage (>80%) of LC neurons respond to elevated
CO
2
/H
+
with an increased firing rate (Elam et al., 1981;
http://dx.doi.org/10.1016/j.neuroscience.2016.03.036
0306-4522/Ó2016 IBRO. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. Address: Dept of Neuroscience, Cell Biology
and Physiology, Wright State University, 3640 Colonel Glenn High-
way, Dayton, OH 45435, USA. Tel: +1-937-775-2288; fax: +1-937-
775-3391.
E-mail address: robert.putnam@wright.edu (R. W. Putnam).
Abbreviations: aCSF, artificial cerebral spinal fluid; Amb, ambiguus
nucleus; AP, area postrema; Bo
¨tz, Bo
¨tzinger complex; Cereb,
cerebellum; CI, chemosensitivity index; cLC, contralateral LC; cNTS,
caudal nucleus tractus solitarius; CVL, caudoventrolateral reticular
nucleus; dHyp, dorsomedial hypothalamus; dlPAG, periaqueductal
gray dorsolateral portion; DR, dorsal raphe; EGTA, ethylene glycol
tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfo
nic acid; iNTS, intermediate nucleus tractus solitarius; IO, inferior olive;
KF, Ko
¨lliker-Fuse; LC, locus coeruleus; lHyp, lateral hypothalamus;
lPAG, periaqueductal gray lateral portion; LRtPC/LRt, lateral reticular
nucleus, parvicellular part/lateral reticular nucleus; LY, Lucifer Yellow;
MR, medullary raphe; NTS, nucleus tractus solitarius; PAG,
periaqueductal gray; PB, parabrachial nucleus; PBS, phosphate-
buffered saline; pHyp, posterior hypothalamus; Pre-Bo
¨tz, Pre-
Bo
¨tzinger complex; Py, pyramidal tract; RTN, retrotrapezoid nucleus;
RVL, rostroventrolateral reticular nucleus; SNB, synaptic blockade
medium; sp5, spinal trigeminal tract; SpVe/Mve, spinal vestibular
nucleus/medial vestibular nucleus; SubC, subcoeruleus region;
SubCA, subcoeruleus nucleus alpha portion; SubCD, subcoeruleus
nucleus dorsal portion; SubCV, subcoeruleus nucleus ventral portion;
vlPAG, periaqueductal gray ventrolateral portion.
Neuroscience 324 (2016) 446–468
446

Pineda and Aghajanian, 1997; Filosa et al., 2002), focal
acidification of the LC increases ventilation (Coates et al.,
1993) and lesioning of a substantial portion of the LC
reduces the ventilatory response to inspired CO
2
(Li and
Nattie, 2006; Biancardi et al., 2008). Interestingly, there is
a marked developmental change in the intrinsic chemo-
sensitivity of LC neurons. In LC neurons from neonates
younger than P10, a high percentage of neurons are CO
2
sensitive (70–80%) and the chemosensitivity index (CI), a
measure of the magnitude of the neuronal chemosensitive
response, is about 235%; whereas in LC neurons from
older neonates (>P10), a high percentage of neurons
are also activated by hypercapnia, but the magnitude of
the response is markedly lower (CI of 125%) (Hartzler
et al., 2008; Nichols et al., 2008; Gargaglioni et al., 2010).
Compared to other regions, most notably the retrotrape-
zoid nucleus (RTN) where the CI is very high at 300%
(determined at 10% CO
2
;Putnam et al., 2004), the CI of
LC neurons from young neonates is fairly large 180–
220% (measured at 7.5%, 10% and 15% CO
2
; Putnam,
unpublished data) while it is very low in LC neurons from
older neonates (115–135% measured at 7.5%, 10% and
15% CO
2
; Putnam, unpublished data). These studies indi-
cate that around 10 days after birth, LC neurons undergo a
major reduction in chemosensitivity with the percentage of
intrinsically chemosensitive neurons and the magnitude of
their response both decreasing dramatically.
As to the NTS, this receives inputs from peripheral
chemoreceptors and lung stretch receptors (Donoghue
et al., 1984; Bonham and McCrimmon, 1990; Mifflin,
1992; Koshiya and Guyenet, 1996) and it is suggested
to be an important site of ventilatory and cardiovascular
control (Barraco et al., 1990; Mifflin, 1992). About
40–50% of NTS neurons depolarize and/or increase their
firing rate upon exposure to hypercapnia (at levels of 7%,
10% and 15% CO
2
(Dean et al., 1989, 1990; Huang et al.,
1997; Mulkey et al., 2004), the magnitude of this
response, as determined by the CI, is about half that of
neurons from the RTN, or about 155–175% (measured
at 15% CO
2
;Conrad et al., 2009), and the chemosensitive
response of these neurons does not change with develop-
ment (Conrad et al., 2009; Nichols et al., 2009). Further,
ventilation is increased when the NTS-especially the cau-
dal part (cNTS) near the area postrema (AP)-is focally
acidified (Coates et al., 1993; Nattie, 2001; Nattie and
Li, 2002) and chemical lesioning or synaptic blockade of
the NTS causes a reduction in basal ventilation and in res-
piratory chemosensitivity to CO
2
(Berger and Cooney,
1982; Nattie and Li, 2002, 2008; although see Favero
et al., 2011). These studies strongly suggest that the
NTS, and especially the cNTS, is involved in central
chemosensitivity and central ventilatory control (Dean
and Putnam, 2010).
Although much is known about these nuclei, central
chemosensitivity appears to involve a complex network
that includes neurons from the LC and cNTS, as well as
many other brainstem areas. This network is poorly
understood, including the roles of the various areas, the
interconnections and the functional communication
between these areas. Also, almost all previous studies
on the nature of the network have involved studies of
adults and thus very little is known about the
development of the central respiratory network.
It is noteworthy that the neuronal pathways we are
studying in neonates play significant roles in functions
others than the control of breathing. Considerable
evidence has accumulated to show that highly emotional
events help in the consolidation of memories (McGaugh,
2000). It appears that epinephrine or other stress-
related chemicals enhance memory consolidation in part
through activating NTS neurons which ultimately results
in ß-adrenergic activation of the basolateral amygdala
(Roozendaal et al., 1999; Clayton and Williams, 2000;
Garcia-Medina and Miranda, 2013; Mello-Carpes and
Izquierdo, 2013). One pathway of excitation from the
NTS to the amygdala that has been proposed involves
projections from the NTS, to the Paragigantocellularis
nucleus, to the LC, and ultimately to the amygdala
(Clayton and Williams, 2000; Mello-Carpes and
Izquierdo, 2013), although the possibility of direct neu-
ronal connections between the NTS and the LC (Van
Bockstaele et al., 1999; Clayton and Williams, 2000)
and the NTS and the basolateral amygdala have been
suggested (Roozendaal et al., 1999). These various path-
ways have been shown to be especially critical to the con-
solidation of memories associated with object recognition
(Mello-Carpes and Izquierdo, 2013) and aversive condi-
tions (Clayton and Williams, 2000). There has been little
study of the development of this memory consolidation
pathway. Nirogi et al. (2012) showed that the consolida-
tion of object recognition memories was markedly less
in juvenile vs. adult rats. They showed that the levels of
norepinephrine were lower in juvenile vs. adult rats but
that the object recognition performance of juvenile rats
could be improved with yohimbine, an aadrenergic recep-
tor antagonist (Nirogi et al., 2012). These data suggest
that the anatomical pathway for memory consolidation is
intact in juveniles. Our current anatomical studies with
neonates are likely to contribute to our understanding of
the development of these important memory consolida-
tion pathways, especially given our focus on the projec-
tions associated with NTS and LC neurons.
Therefore, in this study, we have focused on the LC
and the cNTS of neonatal rats, using retrograde labeling
to identify possible neural connections between these
two areas and other known chemosensitive regions. We
further used whole-cell and loose patch clamping of
retrogradely labeled neurons to test the firing rate
response of neurons to hypercapnia in order to verify
whether neurons projecting from LC to cNTS and from
cNTS to LC could carry chemosensitive information.
EXPERIMENTAL PROCEDURES
Animals
Mixed sex neonatal Sprague–Dawley rats, age between
P7 and P10, were used for microinjection surgeries. The
mothers had free access to water and food and were
housed in a temperature-controlled room at 24–26 °C,
with a 12:12-h light:dark cycle (lights on at 7:00 AM).
The pups stayed together with their mother during all
L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 447

pre- and post-operative periods. All experiments were
done between 8 am and 5 pm. The study was in
compliance with the guidelines of and it was approved
by the Wright State University Institutional Animal Care
and Use Committee. Wright State University is
accredited by AAALAC and is covered by NIH
Assurance (no. A3632-01).
Surgery
For LC surgeries, the surgical procedures were
performed under anesthesia with isofluorane (5%
inhaled to induce anesthesia and 2% inhaled to
maintain anesthesia). For cNTS surgeries, the rats were
anesthetized with ketamine (75 mg/kg I.P.) + xylasine
(5 mg/kg) (0.1 mL/20 g). Subcutaneous bupivicaine
(0.15 mL) was given for the local anesthesia, and
subcutaneous injections of the analgesic agent
carprofen (0.5 mg/ml; vol: 0.1 mL/10 g) were made
before and 12 h after the surgical procedure.
For microinjections of rhodamine beads, the pup was
fixed to a stereotaxic frame (David Kopf, model 900,
Tujunga, CA, USA adapted for neonates) and the skin
was sterilized with povidine solution. Then specific
coordinates were used to locate the LC (antero-
posterior = 3.7 mm; lateral = 1.1 mm from sagittal
suture; dorso-ventral = 4.0 mm deep from the skull).
For cNTS location, the animal’s head was angled 45°
(nose directed downward), an incision with a scalpel in
the neck region was done followed by removing a piece
of the occipital bone as well as the membranes covering
the NTS region, making it possible to view the calamus
scriptorius, used as reference for the coordinates
(lateral = 0.1 mm; dorso-ventral = 0.3 mm). Coordinates
were based on a stereotaxic atlas for rats (Paxinos and
Watson, 1997). A dental injection needle (Mizzy, 200 lm
OD) connected to a 1-lL Hamilton syringe by PE-10
catheter was inserted into the LC or cNTS for 70 lLor
50 lL microinjection of rhodamine beads solution, respec-
tively (Lumafluor, Inc. red retrobeads, St Louis, MO, USA)
and 5 min were allowed to prevent leakage of rhodamine
beads before the injection needle was removed. All
injections were performed manually and the microinjec-
tion volume was chosen based on pilot experiments. At
the end of the surgical procedure, the animal’s skin was
held with surgical glue (Vetbond, Tissue Adhesive) and
cleaned with povidine solution.
Slice preparation
Between 7 to 10 days after the injection of the fluorescent
dye, the labeled rats were then anesthetized with 100%
CO
2
and rapidly decapitated. The brain was removed
and placed in 4–6 °C artificial cerebral spinal fluid
(aCSF) solution equilibrated in 95% O
2
/5% CO
2
(see
Solutions for composition). Coronal slices (300 lm),
extending rostrally from the cNTS (near the AP) to the
hypothalamus were prepared using a PelcoVibratome
1000. Newly prepared slices were either rapidly
transferred into 4% paraformaldehyde solution (see
Solutions) and fixed for 3 days in the refrigerator at 4 °C
for anatomy studies or allowed to equilibrate for 1hr
at room temperature in aCSF equilibrated with 95%
O
2
/5% CO
2
for electrophysiology studies.
Anatomy
After fixation, slices to be used for anatomy studies were
washed in phosphate-buffered saline (PBS, see
Solutions) and placed in a Plexiglas chamber on the
stage of a Nikon Eclipse Ti-S Inverted Light Microscope.
The slice was bathed in a PBS solution and held in
place with a nylon grid. Brightfield illumination was used
to identify the edges of the slice; some references, such
as the central canal (CC), fourth ventricle and
cerebellum (Cereb), were used to identify the
rostrocaudal level and their position in relation to
bregma and also identify various regions within the slice
(based on landmarks from Paxinos and Watson, 1997).
The same region was then studied using epifluorescence.
Rhodamine-labeled neurons were excited at 540–580 nm
and emitted light collected at 600–660 nm using a Nikon
Y-2 E/C fluorescence filter and a 75-W xenon arc lamp.
Emitted fluorescence was visualized using a CoolSNAP
HQ2 digital camera (Photometrics) and Metafluor 4.6r5
software (Universal Imaging) and the fluorescence
images saved to a Gateway 2000 E-3100 computer. Ret-
rogradely labeled slices were first examined at lower
objective (4) and rhodamine-labeled terminal fields were
identified. The regions in which these fields occurred were
verified more accurately using brightfield microscopy,
supported by anatomical markers from Paxinos and
Watson (1997). Such rhodamine-labeled terminal fields
were found in many regions of the brain (Figs. 1 and 2).
The degree of labeling was estimated more precisely by
studying these rhodamine-labeled fields at higher objec-
tive (10for labeling drawings with red dots and in some
instances 20to verify that the labeling was in fact
rhodamine-labeled neurons) using fluorescence micro-
scopy to estimate the number of rhodamine-labeled neu-
ronal cell bodies.
All slices of all animals, from cNTS to the
hypothalamus were examined on the microscope by
only one trained person for retrograde labeling location.
All rhodamine fluorescence observed in each slice’s
region was immediately and carefully marked on
schematic drawings made from Paxinos and Watson
(1997) in order to preserve the anatomical locations of ret-
rograde labeling within the brain.
Electrophysiology
Slices to be used in electrophysiological studies were
placed in a superfusion chamber and held in place with
a nylon grid. Slices were superfused at a rate of
3 mL/min with aCSF or SNB (synaptic blockade
medium) (see Solutions) solution at 35 °C. Glass
electrodes were made from thin-walled borosilicate
glass (outer diameter 1.5 mm, inner diameter 1.12 mm)
pulled to a tip resistance of 3–5 MXwith a Narishige
Model PC-10 pipette puller. The bath was grounded with
an Ag–AgCl
2
wire. Pipettes were filled with pipette filling
solution (see Solutions).
448 L. T. Lopes et al. / Neuroscience 324 (2016) 446–468

LC and cNTS neurons were visualized using an
upright microscope (Nikon FN1) with a 60water-
immersion objective. Neurons retrogradely labeled with
rhodamine beads were visualized using identical
excitation/emission wavelengths as described above
(see Anatomy). Using NIS Elements AR 3.0 software
(Nikon), rhodamine fluorescence was matched to
healthy neurons visualized without fluorescence and
these neurons were targeted for patching.
Electrophysiological recordings were done using an
Axopatch 200B amplifier and analyzed using pCLAMP
software version 10. The pipette was brought to the
surface of a neuron under positive pressure to prevent
the tip from plugging and then a gentle suction was
applied to the pipette until a 1GXseal was obtained.
For cNTS neurons, after the GXseal was established,
a rapid negative pressure was applied to establish a
whole-cell configuration. Such neurons were current
clamped and membrane potential was measured. These
neurons showed the intracellular voltage changes
associated with action potentials and could be used to
determine the firing rate as well. In LC neurons, the cell-
attached patch mode was used to record spontaneous
firing at a holding potential of 0 mV under voltage
clamp. The GXseal in the cell-attached patch gave a
sufficient signal-noise ratio that extracellular currents
were clearly visible and thus firing rate could be
determined from these currents.
For all neurons, recordings began when an initial
stable firing rate was obtained and the effect of
hypercapnia on firing rate was studied by switching the
perfusate (standard aCSF or SNB solution) bubbled with
5% CO
2
to the same perfusate bubbled with 15% CO
2
.
We chose to use this high level of hypercapnia (15%
CO
2
) since our primary aim in these initial
electrophysiology studies was to determine whether
neurons were chemosensitive or not. We have
previously shown with cNTS neurons that about
40–50% of cNTS neurons from neonatal rats will not
change their firing rate in response to this level of
hypercapnia (non-chemosensitive neurons) and about
40–50% will increase their firing rate in response to
hypercapnia (chemosensitive neurons), while the firing
rate in the remaining neurons is reduced upon exposure
to 15% CO
2
(Conrad et al., 2009). Similarly, 15% CO
2
results in an increased firing rate in 70–80% of LC neu-
rons from neonatal rats with the remainder not responding
to hypercapnia (Li and Putnam, 2013). Both cNTS and LC
neurons also respond with increased firing rate to hyper-
capnia of 10% and 7–8% (Dean et al., 1990; Oyamada
et al., 1998; Li and Putnam, 2013) and LC neurons
respond with a decreased firing rate in response to
hypocapnia (2.5% CO
2
)(Li and Putnam, 2013). Thus
the electrophysiology experiments as designed here allow
for the differentiation of whether a neuron is chemosensi-
tive or not.
BA
CD
03.11-amgerB08.31-amgerB
Bregma -7.80Bregma -9.80
AP
sp5
Amb/RVL/
CVL IO
Py
cNTS
(im)
dorsal
Raphé
PAG
RTN
cNTS
(im)
facial
MVePC
LC
PB
Raphé
pallidus
Py
Olivary
SubC
Fig. 1. Schematic drawings of four different slices showing the location of retrogradely labeled neurons (red dots) in different brain regions from
neonatal rats injected with rhodamine beads into the right side of the LC. Note that the microinjection site of rhodamine beads is represented in
schema 3C on LC right side. The schematic drawings are taken from Paxinos and Watson (1997). Each red dot represents a distinct neuron loaded
with fluorescent rhodamine. Amb/RVL/CVL, ambiguus nucleus/rostroventrolateral reticular nucleus/caudoventrolateral reticular nucleus; AP, area
postrema; cNTS, caudal nucleus tractus solitarius; iNTS, intermediate nucleus tractus solitarius; IO, inferior olive; LC, locus coeruleus; MVePC,
medial vestibular nucleus, parvicellular part; PAG, periaqueductal gray; PB, parabrachial nucleus; Py, pyramidal tract; RTN, retrotrapezoid nucleus;
SubC, subcoeruleus; sp5, spinal trigeminal tract.
L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 449

After the maximum effect of hypercapnia on firing rate
was reached, the perfusate bubbled with 15% CO
2
was
changed back to 5% CO
2
, and the firing rate also
returned to the initial baseline, indicating that the
neurons recorded were healthy and the effect of
hypercapnia was reversible. For some neurons, the
effect of hypercapnia was tested in both aCSF and SNB
solution. To assure that we were studying a retrogradely
labeled neuron, the pipette solution contained 2%
Lucifer Yellow (LY). For whole-cell configuration, it took
at least 30 min to complete an experiment and this was
sufficient for the neuron to be well loaded with LY. For
cell-attached configuration, after recording, a rapid
negative suction was applied to get into whole-cell mode
and the neuron was allowed to load with LY for at least
20 min. At the end of all electrophysiology experiments,
slices were fixed in 4% paraformaldehyde for later
confocal imaging to identify rhodamine and LY labeled
neurons. All electrophysiology experiments were
performed at about 35 °C.
Confocal microscopy
Fixed slices were washed 3for 15 min each in 0.1 M
PBS (see Solutions). The washed slice was placed on a
glass slide and covered with Vectashield Mounting
Medium designed for fluorescence (Vector
Laboratories). The slice was then covered with a cover
slip. Fluorescence images were collected with an
Olympus FV300 Confocal Microscope (Olympus
Corporation) with a 60oil objective. Rhodamine bead
fluorescence was visualized with excitation light of
568 nm (from a krypton/argon combination laser) and
LY fluorescence was visualized with excitation light of
488 nm (from a krypton/argon combination laser). Digital
images were collected as Z stacks (0.5 lm resolution).
The image files were managed with Fluoview software
(Olympus Corporation). All confocal images shown are
merged Z stack images to show all the rhodamine
beads within a soma.
Solutions
aCSF was composed of (in mM): 3 KCl, 124 NaCl, 1.3
MgSO
4
, 26 NaHCO
3
, 1.24 NaH
2
PO
4
, 10 glucose, and
2.4 CaCl
2
, equilibrated with 95% O
2
/5% CO
2
gas, pH
7.45. SNB was the same for aCSF except for (in mM):
121 NaCl, 11.4 MgSO
4
, and 0.2 CaCl
2
, plus
carbenoxolone (CAR; 100 lM) to block gap junctions.
Hypercapnic solutions were identical to aCSF and SNB
solutions except for equilibration with 85% O
2
/15% CO
2
gas, pH 6.9–7.0. Paraformaldehyde solutions were
A
C
B
D
Bregma -13.68 Bregma -11.96
Bregma -9.68 Bregma -4.30
Hypothalamus
AP
NTS
Amb/RVL/
CVL Py Raphé
pallidus
Amb/RVL/
Bötz
IO
Raphé
obscurus
sp5
sp5
B4
IRt
SubC
LC
Facial
A5 Py
Fig. 2. Schematic drawings of four different slices showing the location of retrogradely labeled neurons (red dots) in different brain regions from
neonatal rats injected with rhodamine beads into cNTS. Note that the microinjection site of rhodamine beads is represented in schema 4A. The
schematic drawings are taken from Paxinos and Watson (1997). Each red dot represents a distinct neuron loaded with fluorescent rhodamine. Amb/
RVL/CVL, ambiguus nucleus/rostroventrolateral reticular nucleus/caudoventrolateral reticular nucleus; AP, area postrema; B4, B4 serotonin cells;
Bo
¨tz, Bo
¨tzinger complex; IO, inferior olive; IRt, intermediate reticular nucleus; LC, locus coeruleus; cNTS, caudal nucleus tractus solitarius; Py,
pyramidal tract; SubC, subcoeruleus; sp5, spinal trigeminal tract.
450 L. T. Lopes et al. / Neuroscience 324 (2016) 446–468

freshly diluted 1:1 from an 8% paraformaldehyde stock
into 100 mM PBS solution, which contained (in mM):
137 NaCl, 2.7 KCl, 4.3 Na
2
HPO
4
, and 1.47 NaH
2
PO
4
,
pH 7.4. The whole-cell pipette filling solution contained
(in mM): 130 K-gluconate, 0.4 EGTA, 1 MgCl
2
, 0.3 GTP,
2 ATP, and 10 HEPES, plus 2% LY. The pipette filling
solution pH was adjusted to 7.35 using KOH.
Data analysis and statistics
For anatomy study, the labeling intensity was determined
as follows: 0–1 neuron identified was assigned the
number ‘‘0”. In the same way, 2–5 neurons were
designated as ‘‘1”; 6–10 neurons designated as ‘‘2”; and
more than 10 neurons designated as ‘‘3”; three animals
per group were scored. For the pattern of labeling, all
regions with high intensity of labeling (number 2 or 3)
after injections into the LC or into the cNTS, were
assessed to see if these areas have been associated
with ventilatory control. Thus, each heavily loaded area
was carefully evaluated for the location of the retrograde
labeling within the nucleus. From the total amount of
labeling within the region, the labeling in the nucleus
was subdivided into dorsal, lateral and/or ventral
portions, as well as the location rostral-caudal relative to
bregma.
The firing rate of rhodamine-loaded LC and cNTS
neurons was calculated as the integrated firing rate over
a 10-s bin using Clampfit 10.3 software. Thus, the
number of action potentials was determined for each
10-s period and plotted as the number of firings per 10 s
vs. time plots. For each neuron, the average firing rate
over a 1-min period was determined either in 5% CO
2
or
15% CO
2
, and plotted as firing rate per s (Hz). All
values were expressed as mean ± SEM, when we had
at least three neurons. Significance was set as P60.05
and significant differences were determined using paired
T-tests.
RESULTS
Anatomy
Microinjection of rhodamine beads into the LC. To
assess specificity of retrograde labeling from the LC, the
results were divided into four groups (Table 1): (1) LC
+: microinjection of rhodamine beads centered into the
LC (Fig. 3A); (2) LC Miss: injection site centered
ventrally and/or laterally from the LC located in the
Table 1. Numbers of retrogradely labeled neurons in various brainstem regions from rhodamine injections into either the LC or the cNTS
Scoring: ‘‘0”: 0–1 neuron identified; ‘‘1”: 2–5 neurons identified. The numbers in black represents a weak labeling pattern. ‘‘2”: 6–10 neurons identified. The numbers in green
represents a median labeling pattern. ‘‘3”: >10 neurons identified. The numbers in red represents a strong labeling pattern. Highlighted areas in yellow represent regions
involved in ventilatory control. N= 3; ND, not determined.
L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 451

A
B
4th ventricle
midline
4th ventricle
midline
4th ventricle
Cerebellum
Cerebellum
4th ventricle
midline
C
D
Brightfield Brightfield and Fluorescent Fluorescent
Fig. 3. Microinjection of rhodamine beads in the LC of neonatal rats. The left column shows brightfield images, the middle column shows brightfield
and fluorescence imagens and the right column shows fluorescence images of a pontine slice containing the LC. For orientation, the floor of the 4th
ventricle and the midline of the slice are indicated (white arrows) and the location of the LC is outlined by the white dotted ellipse. (A) LC Hit. Note in
the fluorescence image that the rhodamine beads (red fluorescence) lie entirely within the LC. (B) LC-Miss. Note in the fluorescent image that the
rhodamine beads lie entirely out, ventrally of the LC. (C) Peri-LC. Note in the fluorescent image that the rhodamine beads do not lie within the LC but
in an adjacent area abutting the LC, or the peri-LC. (D) LC and Cerebellum Hit. Note in the fluorescent image that the rhodamine beads lie in the LC
as well as within the cerebellum. The calibration bar in each image represents 300 lm.
452 L. T. Lopes et al. / Neuroscience 324 (2016) 446–468

B. Bregma -7.80 mm
DMPAG
9% DLPAG
7%
LPAG
37%
VLPAG
47%
ROb
21%
RPa
63%
C. Bregma -12.80 mm
D. Bregma -9.80 mm
RIp
4%
RMg
12%
A. Bregma -13.80 mm
cNTS
62%
RTN
57%
E. Bregma -10.52 mm
Fig. 4. Schematic drawings of different brain regions showing the pattern of location of retrogradely labeled neurons within certain nuclei from neonatal
rats injected with rhodamine beads into LC. The schematic drawings are taken from Paxinos and Watson (1997). (A) cNTS: caudal nucleus tractus
solitaries; 162 neurons with rhodamine-loaded soma were counted in 12 slices between Bregma 13.80 and 14.40 mm; of these, 62% neurons were
localized to the dorsolateral, commissural and medial portion of the cNTS (red circle). (B) PAG: periaqueductal gray: 90 neurons with rhodamine-loaded
soma were counted in 18 slices between Bregma 7.30 and 8.80 mm; of these, 9% were localized to the dorsomedial portion (dmPAG), 7% to the
dorsolateral portion (dlPAG), 37% to the lateral portion (lPAG) and 47% to the ventrolateral portion (vlPAG) (area defined by dashed red line). (C) RPa:
raphe pallidus nucleus, ROb: raphe obscurus nucleus; 65 neurons with rhodamine-loaded soma were counted in 14 slices between Bregma 9.72 and
14.16 mm; of these 63% were in the raphe pallidus nucleus and 21% of these were in the raphe obscurus nucleus. (D) RMg: raphe magnus nucleus,
RIp: raphe interpositus nucleus; 12 neurons with rhodamine-loaded soma were counted in 12 slices between Bregma 9.6 and 12.12 mm; of these
4% were localized to the raphe interpositus and 12% to the raphe magnus. (E) RTN: retrotrapezoid nucleus; 37 neurons with rhodamine-loaded soma
were counted in nine slices between Bregma 10.44 and 10.88 mm; of these 57% were in the caudal region of the RTN (red circle). (F): Pre-Bo
¨tz:
Pre-Bo
¨tzinger complex; 20 neurons with rhodamine-loaded soma were counted in six slices between Bregma 12.30 and 12.84 mm; of these 70%
were in the dorsal part of the Pre-Bo
¨tz Complex (red circle). (G) Bo
¨tz: Bo
¨tzinger complex; 27 neurons with rhodamine-loaded soma were counted in six
slices between Bregma 11.96 and 12.48 mm; of these 70% were in the dorsal part of the Bo
¨tz Complex (red circle). (H) lHyp: lateral hypothalamus;
38 neurons with rhodamine-loaded soma were counted in 12 slices between Bregma 1.20 and 4.56 mm; of these 70% were in the dorsal part of the
Bo
¨tz Complex (red circle). (I) cLC: contralateral LC; 19 neurons with rhodamine-loaded soma were counted in 12 slices between Bregma 8.88 and
10.04 mm; of these 53% were in the dorsal part of the contralateral LC (red circle).
L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 453

medial vestibular nucleus, magnocellular part (MVeMC),
dorsomedial spinal trigeminal nucleus (DMsp5), or
peritrigeminal zone/parvicellular reticular nucleus, alpha
part (P5/PCRtA) (Fig. 3B); (3) Peri LC: microinjection of
rhodamine beads located in the region surrounding the
LC (Fig. 3C); and (4) LC+Cereb: microinjection placed
within the LC, but with vestiges of rhodamine in the
Cereb (Fig. 3D).
LC+ group: substantial numbers of rhodamine-
labeled cells (See Table 1, ‘‘3” in red) were located in a
variety of regions thought to be involved in ventilatory
control (yellow rows), including the periaqueductal gray
(PAG), contralateral LC (cLC), subcoeruleus region
(SubC), parabrachial nucleus (PB), Ko
¨lliker-Fuse
nucleus (KF), cNTS, medullary raphe (MR) and RTN.
Moderate labeling (see Table 1, ‘‘2” in green) was found
in other chemosensitive regions such as the
dorsomedial and lateral hypothalamus (dHyp and lHyp
respectively), A5 region, Amb/RVL/CVL region
(ambiguus nucleus/rostroventrolateral reticular nucleu
s/caudoventrolateral reticular nucleus), Pre-Bo
¨tzinger
and Bo
¨tzinger complex (Pre-Bo
¨tz/Bo
¨tz). We found
strong labeling in some non-chemosensitive areas such
as inferior olive (IO), lateral reticular nucleus,
parvicellular part/lateral reticular nucleus (LRtPC/LRt)
and pyramidal tract (Py), and moderate labeling in non-
chemosensitive areas such as the Facial Nucleus,
Paragigantocellular nucleus, intermediate NTS (iNTS)
and spinal trigeminal tract (sp5). Areas with lower
intensity of labeling with scattered cells (see Table 1,
‘‘1” or ‘‘0” in black) were found only in non-
chemosensitive areas (Table 1), including: AP, posterior
hypothalamus (pHyp), DR, IO, lateral reticular nucleus,
LRtPC/LRt, Py, and spinal vestibular nucleus/medial
vestibular nucleus (SpVe/Mve).
LC+Cereb: the pattern of retrograde labeling was
very similar to the LC+ group, as expected (Table 1).
Peri LC: retrograde labeling was moderate, with a
reduction for some areas compared with the LC+
group, but still present in most regions (Table 1).
LC Miss: there was almost no retrograde labeling in
the regions examined (Table 1).
These findings suggest that the neuronal projections
seen with retrograde labeling are largely specific to the
LC region.
Pattern of retrograde labeling: afferent projections to
LC. We focused our analysis of retrograde labeling
location in nuclei in which moderate (2) to intense (3)
labeling were observed after rhodamine bead injection
into the LC. Only animals belonging to the LC+ group
and the LC Miss group (control) were considered.
Among the chemosensitive areas with such
retrograde labeling is the cNTS. In the cNTS, the
dorsolateral, commissural and medial portion of the
nucleus was the region with majority neurons loaded
with rhodamine (62%), while 38% of labeled neurons
were observed in the interstitial, intermediate, ventral
and ventrolateral regions (Fig. 4A).
G. Bregma -11.96 mm
Botz
70%
F. Bregma -12.30 mm
Pre-Botz
70%
lHyp
76%
H. Bregma -1.80 mm I. Bregma -10.04 mm
cLC
53%
Fig. 4 (continued)
454 L. T. Lopes et al. / Neuroscience 324 (2016) 446–468

The second region with higher intensity of retrograde
labeling was the PAG. Most of the labeled neurons were
present in the ventrolateral and lateral PAG, about 47%
and 37%, respectively (Fig. 4B). As for the other
subdivisions, in the dorsomedial and dorsolateral PAG
less than 10% of labeling was observed. Fig. 4B also
shows that approximately 64% of all PAG labeling was
found near the aqueduct to the medial region.
The MR was the third region with a large number of
retrogadely labeled neurons. Of the loaded neurons in
this region, 63% were in the raphe pallidus, 21% were
in the raphe obscurus, 12% in the raphe magnus and
only 4% were in the raphe interpositus nucleus (RIp)
(Fig. 4C, D). In the RTN, 57% of the labeled neurons
occurred in the caudal region of the nucleus, specifically
in the lateral portion (Fig. 4E).
Other regions that have moderate labeling patterns
were the lHyp, Pre-Bo
¨tz and Bo
¨tz region. In the Pre-
Bo
¨tz and Bo
¨tz region, most of the labeled neurons were
located in the dorsal portion, as shown in Fig. 4F, G.
For the lHyp, 76% of the labeled neurons were located
in the ventral portion (Fig. 4H). However, this pattern
was only observed for more rostral coordinates, since
for the caudal region of lHyp a more homogeneous
distribution occurred. Also in the cLC, 53% of the
labeled cells were found in the dorsal portion of the
nucleus, followed by the ventral and middle portion,
26% and 21% respectively (Fig. 4I).
It was observed that other nuclei, such as dHyp,
SubC, PB, KF, A5 region and AMB/RVL/CVL had
scattered projections to LC but no particular loading
pattern was noted. Also, for the LC Miss group none of
the patterns observed in the LC+ group were found.
Microinjection of rhodamine beads into the cNTS. The
results were divided into two groups: (1) cNTS+:
microinjection of rhodamine beads centered into the
cNTS (Fig. 5A); and (2) cNTS Miss: injection site
located out of the cNTS, placed in the cuneate nucleus
and gracile nucleus (Fig. 5B).
cNTS+: chemosensitive regions containing
rhodamine fluorescence with high density (Table 1, ‘‘3”
in red) were found only in the SubC and A5 regions. We
found strong labeling also in the non-chemosensitive
iNTS. Moderate labeling (Table 1, ‘‘2” in green) was
observed in chemosensitive areas such as the dHyp
and lHyp, PAG, LC, KF, Amb/RVL/CVL, Pre-Bo
¨tz/Bo
¨tz
and RTN. Moderate labeling was observed also in two
non-chemosensitive areas, the LRtPC/LRt and the sp5.
Chemosensitive areas with lower intensity of labeling
with scattered or individual cells (Table 1, ‘‘0” or ‘‘1” in
black) were found in PB and MR. The neurons
CC
Hy Hy
NTSNTS
DMN
DMN
AP
Hy Hy
CC
NTSNTS
DMN
DMN
AP
CC
Hy Hy
NTSNTS
DMN
DMN
AP
CC
Hy Hy
NTSNTS
DMN
DMN
AP
Hy Hy
NTSNTS
DMN
DMN
AP
CC
CC
Hy Hy
NTSNTS
DMN
DMN
AP
Brightfield Brightfield and Fluorescent Fluorescent
A
B
Fig. 5. Microinjection of rhodamine beads into the cNTS of neonatal rats. The left column shows brightfield images, the middle column shows
brightfield and fluorescence imagens and the right column shows fluorescence images of a medullary slice containing the cNTS. For orientation, the
general area of the hypoglossal (Hy) nucleus is marked, and the central canal (CC), the area postrema (AP), the dorsal motor nucleus of the vagus
(DMN) and the NTS are delineated by white dotted lines. (A) NTS Hit. Note in the fluorescence image that the rhodamine beads (red fluorescence)
lie entirely within the NTS. (B) NTS Miss. Note in the fluorescence image that the rhodamine beads lie entirely outside of the NTS. The calibration
bar in each image represents 300 lm.
L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 455

D. Bregma -7.80 mm
DMPAG
15% DLPAG
32%
LPAG
21%
VLPAG
32%
A. Bregma -10.80, 10.30 and 9.80 mm
RTN
~100%
E. Bregma -10.52 mm
A5
72%
B. Bregma -9.68 mm
SubCA
12%
SubCD
39%
SubCV
49%
C. Bregma -10.04 mm
LC
51%
Fig. 6. Schematic drawings of different brain regions showing the pattern of location of retrogradely labeled neurons within the nuclei from neonatal
rats injected with rhodamine beads into cNTS. The schematic drawings are taken from Paxinos and Watson (1997). (A) A5 region; 106 neurons with
rhodamine-loaded soma were counted in 24 slices between Bregma 9.00 and 11.28 mm; of these, 72% were localized to the caudal portion of the
A5 region. (B) SubC: subcoeruleus nucleus alpha portion (SubCA), dorsal portion (SubCD) and ventral portion (SubCV); 103 neurons with rhodamine-
loaded soma were counted in 12 slices between Bregma 8.88 and 9.96 mm; of these, 12% were localized to the SubCA region, 39% to the SubCD
region, and 49% to the SubCV region (red circle). (C) LC: locus coeruleus; 58 neurons with rhodamine-loaded soma were counted in 12 slices between
Bregma 8.80 and 10.04 mm; of these, 51% were localized to the dorsal region of the LC (red circle). (D) PAG: periaqueductal gray dorsomedial
portion (dmPAG), dorsolateral portion (dlPAG), lateral portion (lPAG) and ventrolateral portion (vlPAG); 48 neurons with rhodamine-loaded soma were
counted in 18 slices between Bregma 7.30 and 8.80 mm; of these, 15% were localized to the dmPAG region, 32% in the dlPAG region, 21% in the
lPAG region and 32% in the vlPAG region (all areas defined by the dashed red line). (E) RTN: retrotrapezoid nucleus; 30 neurons with rhodamine-
loaded soma were counted in 9 slices between Bregma 10.44 and 10.80 mm; of these,100% were localized tothe caudal region and centralarea of
the RTN (red circle). (F) KF: Ko
¨lliker-Fuse; 41 neurons with rhodamine-loaded soma were counted in six slices between Bregma 8.72 and 8.80 mm;
of these, 83% were localized to the ventral portion of the KF (red circle). (G) Pre-Bo
¨tz: Pre-Bo
¨tzinger complex; 64 neurons with rhodamine-loaded soma
were counted in six slices between Bregma 12.30 and 12.84 mm; of these, 72% were localized to the ventral portion of the Pre-Bo
¨tz (red circle). (H)
Bo
¨tz: Bo
¨tzinger complex; 50 neurons with rhodamine-loaded soma were counted in six slices between Bregma 11.96 and 12.48 mm; of these, 72%
were localized to the ventral portion of the Bo
¨tz (red circle). (I) lHyp: lateral hypothalamus. Sixty-three neurons with rhodamine-loaded soma were
counted in 18 slices between Bregma 1.2 and 4.56 mm; of these, 77% were localized to the ventral portion of the lHyp (red circle).
456 L. T. Lopes et al. / Neuroscience 324 (2016) 446–468

projecting to the NTS from non-chemosensitive areas
were from the perifornical area (PA), pHyp, DR, Facial
nucleus, IO, Py and SpVe/Mve. The neurons projecting
to the NTS from non-chemosensitive areas were the
same that projected to the LC.
cNTS Miss: retrograde labeling was nearly absent in
cNTS miss when compared to when injections were in
cNTS+.
Pattern of retrograde labeling: afferent projections to
cNTS. The A5 region and the SubC region both had a
similar large number of labeled neurons. For the A5
region, most labeled neurons (72%) occurred in the
caudal portion (Fig. 6A). For the SubC region, most
labeled cells were observed in the ventral part (49%),
followed by the dorsal and alpha part, 39% and 12%,
respectively (Fig. 6B).
With respect to the LC, about 51% of all labeled
neurons occurred in the dorsal portion (Fig. 6C). In
addition, 32% of labeled neurons were seen in the
ventral portion followed by the medial portion (17%) of
the LC.
We also studied the labeling pattern in the PAG, RTN,
KF, Pre-Bo
¨tz and Bo
¨tz region, lHyp and dHyp. For the
PAG, most of the loaded cells were present in the
ventrolateral and dorsolateral PAG subdivision, with
32% for each one, following by 21% in the lateral and
15% in the dorsal medial PAG subdivision (Fig. 6D).
Approximately 75% of all PAG labeling was found near
the aqueduct to the medial region (Fig. 6D). In the RTN
nucleus, all labeled neurons were located in the caudal
region of the nucleus, specifically in the central area of
this nucleus, as highlighted in Fig. 6E. For the KF, 83%
of the retrogradely labeled neurons were found in the
ventral portion of the nucleus and the remaining labeled
neurons were widespread in the dorsal and medial
portion (Fig. 6F). A similar pattern of labeling (ventral
prevalence) was present in the Pre-Bo
¨tz and Bo
¨tz
region, since 72% of the rhodamine-labeled cells were
found in this portion of these nuclei (Fig. 6G, H). Finally,
the lHyp was the nucleus of the hypothalamic region
with the most expressive labeling, wherein 77% of all
cells occurred in the ventral portion, followed by the
dHyp (Fig. 6I).
As to Amb/RVL/CVL, labeled neurons were observed
dispersed throughout the whole nucleus.
For the cNTS Miss group none of these patterns noted
in the cNTS+ group were found.
Retrograde labeling and LY. Confocal microscopy
was performed on selected slices from various regions
of the brainstem to confirm that apparent retrograde
H. Bregma -11.96 mm
Botz
72%
G. Bregma -12.30 mm
Pre-Botz
72%
lHyp
77%
I. Bregma -1.80 mm
KF
83%
F. Bregma -8.72 mm
Fig. 6 (continued)
L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 457

labeling was due to rhodamine bead-labeled cell bodies.
Indeed, these regions revealed neuronal cell bodies that
contained rhodamine beads (red fluorescence) in the
cytoplasm surrounding the nucleus, as well as in the
axonal processes of some neurons (Fig. 7), as expected
for a dye that loads retrogradely. After injection of
rhodamine beads into the LC, several of these red-
labeled neurons were visible in the cNTS (Fig. 7A, B;
white arrows). Rhodamine-labeled neurons were also
seen in the region of the RTN (Fig. 7C, white arrow)
and in the PAG, where both loaded soma (white arrows)
and axonal processes (blue arrows) were seen
(Fig. 7D). Similarly, when rhodamine beads were
injected into the cNTS dye-loaded LC neuronal cell
bodies (Fig. 7E, F; white arrows) and axonal processes
(Fig. 7F, blue arrows) were seen.
These rhodamine-labeled cell bodies were also visible
under epifluorescence microscopy during the
electrophysiological experiments and were targeted for
patch-clamp study. To ensure that the retrogradely
labeled neuron was the same that was studied in the
electrophysiology protocol, the patched neuron was
loaded with LY, added into the patch pipette solution.
After the electrophysiological study, the slice was
verified in the confocal microscopy to confirm if the
neuron studied was positive for both rhodamine and LY
labeling. Examples of such neurons from the cNTS and
the LC are shown in Fig. 8. The top panel shows
neurons from the cNTS that were retrogradely labeled
with rhodamine beads (left side; white arrow) that had
been injected into the LC, and the same neuron loaded
with LY (right side; green fluorescence, white arrow)
(Fig. 8A). The neuron indicated by the white arrow is a
retrogradely labeled cNTS neuron that was studied
electrophysiogically and afterward, loaded with LY
(green fluorescence). Another neuron is also visible in
the figure (right panel; blue arrow) which represents a
neuron with retrograde labeling but without the LY
fluorescence, which means that this neuron was not
patched and studied for electrophysiological properties.
cNTS Neurons RTN Neurons
A
B
PAG Neurons
C
D
LC Neurons
E
F
Fig. 7. Confocal images of retrogradely labeled neurons after injection of rhodamine stack images. (A, B) Neuronal cell bodies from the cNTS
loaded with rhodamine beads (red fluorescence) (white arrows) indicating that neurons from the cNTS have afferent projections to the LC. Note
multiple rhodamine beads in each soma indicating heavy retrograde labeling. (C) A neuronal cell body from the area of the RTN loaded with
rhodamine beads (red fluorescence) (white arrow). Note there are several rhodamine beads in the soma. (D) Neuronal cell bodies from the PAG
loaded with rhodamine beads (red fluorescence) (white arrows) indicating that neurons from the PAG have afferent projections to the LC. Note
multiple rhodamine beads in each soma. Also note the presence of rhodamine beads in a neuronal process (blue arrows). (E, F) Neuronal cell
bodies from the LC loaded with rhodamine beads (red fluorescence) (white arrows) indicating that neurons from the LC have afferent projections to
the cNTS. Note multiple rhodamine beads in each soma indicating heavy retrograde labeling. Also note the presence of rhodamine beads in
neuronal processes (blue arrows in F). cNTS, caudal nucleus tractus solitarius; LC, locus coeruleus; PAG, periaqueductal gray; RTN, retrotrapezoid
nucleus. The calibration bar in each image represents 50 lm.
458 L. T. Lopes et al. / Neuroscience 324 (2016) 446–468

In the bottom panel, a retrogradely labeled LC neuron is
shown (left side; red fluorescence, white arrow) that was
labeled with rhodamine beads that had been injected
into the cNTS (Fig. 8B). This same neuron was
patched, studied electrophysiologically and then injected
with LY (right side; yellow fluorescence, white arrow)
(Fig. 8B).
Electrophysiology
Fig. 9A shows that hypercapnic acidosis induced a
striking increase in the firing rate of rhodamine-labeled
cNTS neurons. This effect was also maintained in SNB
solution (Fig. 9B), indicating that this chemosensitive
response of firing rate in rhodamine-labeled cNTS
neurons is intrinsic and not dependent on synaptic
transmission. In four of five patched rhodamine-labeled
cNTS neurons, hypercapnic acidosis in SNB solution
induced depolarization and a significant increase in
firing rate (0.58 ± 0.3 Hz in 5% CO
2
vs. 1.26 ± 0.5 Hz
in 15% CO
2
,n=4, p= 0.04, Fig. 9C). In one neuron,
exposure to hypercapnia caused a hyperpolarization
and reduction in firing rate (0.73 Hz in 5% CO
2
vs.
0.27 Hz in 15% CO
2
,Fig. 9D). This result suggests that
the majority of cNTS neurons with projections to the LC
appear to be intrinsically chemosensitive.
Fig. 10A shows that hypercapnic acidosis led to an
apparent decrease in firing rate of rhodamine-labeled
LC neurons. This effect was also maintained in SNB
solution (Fig. 10B), suggesting that this rhodamine-
labeled LC neuron is intrinsically CO
2
chemosensitive.
In two of the three neurons, the addition of hypercapnia
resulted in a reversible decrease in firing rate (1.67 Hz
in 5% CO
2
vs. 0.94 Hz in 15% CO
2
; 0.19 Hz in 5% CO
2
vs. 0.01 Hz in 15% CO
2,
Fig. 10C). In the third, firing
rate was unchanged or slightly decreased by
hypercapnia (0.4 Hz in 5% CO
2
vs. 0.33 Hz in 15%
CO
2
,Fig. 10C). Fig. 10D demonstrates that the firing
rate dropped from 1.23 Hz to 0.014 Hz by hypercapnia
in the same neuron shown in Fig. 10B. These results
suggest that the LC neurons with projections to the
cNTS may be inhibited by hypercapnia.
cNTS NEURON
LC NEURON
Rhodamine Rhodamine + LY
Rhodamine + LYRhodamine
A
B
Fig. 8. (A) Neurons from the cNTS that have been retrogradely labeled with rhodamine beads injected into the LC. In the left panel is indicated a
cNTS neuronal cell body with numerous rhodamine beads (white arrow). This neuron was patched and studied electrophysiologically and then
loaded with Lucifer Yellow (LY). In the right panel the same neuron seen in the left panel shows extensive loading with LY (green fluorescence) with
several yellow dots that indicate the overlap of the rhodamine beads seen in the left panel with LY (white arrow). Note a separate neuron that is
loaded with rhodamine beads but not with LY (blue arrow). (B) In the left panel is a neuron from the LC that has been retrogradely labeled with
rhodamine beads (white arrow) injected into the cNTS. This neuron was studied electrophysiologically and then loaded with LY, but at the end of the
recording, the neuron was damaged (white arrow). It is clear that the LY-loaded neuron is in the same location as the rhodamine-labeled neuron in
the left panel. All images shown are merged Z stack images. The calibration bar in each image represents 50 lm.
L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 459

Rhodamine-loaded cNTS neuron
Fig. 9. (A) The firing response to hypercapnia (whole-cell patch in current-clamp mode) of a neuron from the cNTS that contains rhodamine beads
retrogradely labeled from the LC, the site of rhodamine bead injection. (A1) The firing rate response to hypercapnic acidotic solution (15%CO
2
)in
the presence of aCSF. (A2) The integrated firing rate (action potentials per 10 s) in normocapnic (5% CO
2
) and hypercapnic (15% CO
2
) solutions.
(B1) The firing rate response of the same neuron as in A to hypercapnic acidotic solution (15% CO
2
) in the presence of synaptic block medium
(SNB). (B2) The integrated firing rate of the same neuron as in A in normocapnic (5% CO
2
) and hypercapnic (15% CO
2
) SNB solution. Note that
hypercapnia reversibly increased the firing rate of this cNTS neuron that projects to the LC even in the presence of SNB solution. (C) Summary of
the effect of hypercapnia in SNB solution on firing rate (Hz: action potentials per s) in four rhodamine-labeled cNTS neurons. (D) Hypercapnia
caused reduction in firing rate in one rhodamine-labeled cNTS neuron.
460 L. T. Lopes et al. / Neuroscience 324 (2016) 446–468

Rhodamine-loaded LC neuron
Fig. 10. (A) The firing response to hypercapnia (loose-cell patch in current-clamp mode) of a neuron from the LC that contains rhodamine beads
retrogradely labeled from the cNTS, the site of rhodamine bead injection. (A1) The firing rate response to hypercapnic acidotic solution (15% CO
2
)in
the presence of aCSF. (A2) The integrated firing rate (action potentials per 10 s) in normocapnic (5% CO
2
) and hypercapnic (15% CO
2
) solutions.
(B1) The firing rate response of the same neuron as in A to hypercapnic acidotic solution (15% CO
2
) in the presence of synaptic block medium
(SNB). (B2) The integrated firing rate of the same neuron as in A in normocapnic (5% CO
2
) and hypercapnic (15% CO
2
) SNB solution. Note that
hypercapnia reversibly decreased the firing rate of this LC neuron that projects to the cNTS even in the presence of SNB solution. (C) Summary of
the effect of hypercapnia in aCSF on firing rate (Hz: action potentials per s) in three rhodamine-labeled LC neurons. (D) The reduction in firing rate
by hypercapnia was maintained in SNB solution in the same rhodamine-labeled LC neuron shown in B.
L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 461

DISCUSSION
In the present study, we used retrograde labeling in
neonatal rat pups to investigate afferent projections from
a variety of brain regions to two chemosensitive areas,
the LC and the cNTS (Nattie and Li, 2009; Nattie,
2011). In addition, we have begun to characterize the
functional connections between these two regions using
electrophysiological techniques. Among the main find-
ings, there are considerable anatomical connections
between numerous chemosensitive sites and at least
some of the connections appear to be capable of trans-
mitting chemosensitive information in neonates as young
as P7–P10. These findings indicate that the central
chemosensitive network is likely to be both anatomically
and functionally complex, even in neonatal rats. Our data
also suggest that pathways believed to be involved in the
consolidation of aversive and object recognition memo-
ries (Clayton and Williams, 2000; Mello-Carpes and
Izquierdo, 2013) may be already present in neonatal rats.
Anatomy results
Afferent projections to the LC. Based on our findings
(Figs. 1 and 4 and Table 1), LC neurons in neonatal
rats receive afferent projections from many regions
involved in cardiorespiratory and autonomic control
including dHyp, lHyp, PAG, cLC, SubC, PB, KF, A5,
Amb/RVL/CVL, Pre-Bo
¨tz/Bo
¨tz, cNTS, MR and RTN. The
results of the present study in neonatal rats agree in
general with a previous study by Cedarbaum and
Aghajanian (1978) performed in adult rats with horserad-
ish peroxidase (HRP) injected into LC. These authors
showed a similar retrograde labeling pattern in hypothala-
mus, PB and NTS. Also, other anatomical studies using
adult rats have described afferents to the LC from neu-
rons of the PB (Luppi et al., 1995), hypothalamic areas
(Conrad and Pfaff, 1976a,b; Saper et al., 1976;
Swanson, 1976, 1977; Swanson and Cowan, 1977;
Luppi et al., 1995; Lee et al., 2005; Reyes et al., 2005),
KF (Luppi et al., 1995), PAG (Ennis et al., 1991; Van
Bockstaele and Aston-Jones, 1992; Luppi et al., 1995;
Odeh and Antal, 2001; Lee et al., 2005) and MR (Sim
and Joseph, 1992).
In relation to PAG, it is important to mention that the
retrograde labeling observed in the LC+ group was
concentrated on the lateral (37%) and ventrolateral
(47%) region. PAG is divided into four longitudinal
columns (dorsomedial, dorsolateral, lateral, and
ventrolateral) that are proposed to have differing
functions (Carrive and Bandler, 1991; Bandler et al.,
1991). Our findings are in agreement with other studies
that showed cell bodies of neurons from adults projecting
from the PAG to the LC are most concentrated within the
ventrolateral column (Morgane and Jacobs, 1979;
Mantyh, 1983; Aston-Jones et al., 1991; Luppi et al.,
1995; Bajic and Proudfit, 1999; Odeh and Antal, 2001).
Likewise, neurons in the ventrolateral column of the
PAG also strongly project to the peri-LC (Aston-Jones
et al., 1991; Luppi et al., 1995; Bajic and Proudfit, 1999).
We also observed a strong labeling (63%) in the raphe
´
pallidus. These projections, though sparse, have been
confirmed through anterograde methods in adult cats
(Bobillier et al., 1976) and rats (Sim and Joseph, 1992)
and also through retrograde methods in adult rats
(Cedarbaum and Aghajanian, 1978; Morgane and
Jacobs, 1979; Luppi et al., 1995). Raphe
´pallidus is
involved in respiratory control in mammals and has exci-
tatory modulation of ventilation (Lalley, 1986; Cao et al.,
2006a,b). Neurotracing studies revealed that this nucleus
projects to spinal phrenic motor neurons (Dobbins and
Feldman, 1994; Hosogai et al., 1998) as well as medullary
phrenic pre-motor neurons (Smith et al., 1989; Lindsey
et al., 1994; Song et al., 2001).
Regarding NTS, connections from the cNTS to the LC
have been demonstrated through anterograde labeling
studies (Van Bockstaele et al., 1999) as well as retro-
grade labeling studies, both in adult rats (Cedarbaum
and Aghajanian, 1978; Clavier, 1979; Aston-Jones
et al., 1991; Luppi et al., 1995; Rampon et al., 1999).
Taken together, these results indicate that afferent projec-
tions to LC neurons in neonatal and adult rats are similar
and involve numerous regions putatively related to the
central chemosensitive network.
It is well known that the dendrites of LC neurons
ramify heavily and selectively in the rostromedial part of
SubC (Aston-Jones et al., 1991; Van Bockstaele et al.,
2001). The innervations of dendrites in peri-LC areas
may have a significant influence on the activity of LC neu-
rons because some studies have shown that LC pro-
cesses may interact directly via electrotonic coupling
(Williams et al., 1991; Huang et al., 1997; Dean et al.,
1997; Veznedaroglu et al., 1998). This could explain
why the peri-LC injected group shows a similar pattern
of retrograde labeling as the LC+ group (Table 1).
Regardless it is clear that the LC is receiving considerable
afferent input from regions involved in the central
chemosensitive network, which could account for the abil-
ity of serotonergic nerve endings (perhaps from the
raphe) to modulate the activity of LC neurons
(Cedarbaum and Aghajanian, 1978; Van Bockstaele,
2000) and is consistent with LC neurons exhibiting a level
of activity that is modulated at respiratory frequency
(Oyamada et al., 1998).
Afferent projections to the cNTS. When rhodamine
was injected into the cNTS, the retrograde labeling
pattern was smaller compared to LC injections. Further,
fewer projections to cNTS were found compared with
the LC afferents (Table 1).
Similar to LC, the cNTS neurons from neonatal rats
receive afferent projections from regions involved in
cardiorespiratory and autonomic control (Figs. 2 and 6
and Table 1), including dHyp, lHyp, PAG, LC, SubC,
KF, A5, Amb/RVL/CVL, Pre-Bo
¨tz/ Bo
¨tz, MR and RTN.
Afferent projections to the cNTS have previously been
observed from the ventral medullary respiratory group,
and specifically from the RVL, Bo
¨tz and Pre-Bo
¨tz (Tan
et al., 2010; Alheid et al., 2011). In addition, projections
from hypothalamus to cNTS have also been shown
(Van der Kooy et al., 1984). Like the LC, most projections
462 L. T. Lopes et al. / Neuroscience 324 (2016) 446–468

to the cNTS observed in adulthood are already estab-
lished in newborns.
The numerous projections from the ventral medullary
respiratory group, including Amb/RVL/CVL, Pre-Bo
¨tz as
well as the RTN strongly suggest that the neonate
cNTS is involved in breathing regulation. In fact, cNTS
has been suggested to be a site for integration of
respiratory, cardiovascular and gastroesophageal
systems that work together to eliminate CO
2
during
acute and chronic respiratory acidosis to restore pH
homeostasis (Dean and Putnam, 2010).
The cNTS Miss group showed little to no retrograde
labeling compared to the group where rhodamine
injections were clearly in the cNTS (cNTS +) (Table 1),
which shows the specificity of the afferent projections for
the cNTS.
Functional connections between the cNTS and
LC. Considerable anatomical connections among
various brainstem regions were found that are believed
to be a part of the central chemosensitive network.
Since these regions are not only involved in breathing
control, it is likely that some of the anatomical
connections are involved in other physiological
functions. For instance, it is well known that the control
of breathing is state dependent, changing with sleep or
anesthesia (Nattie, 2001; Kuwaki, 2010). Orexin has been
proposed to contribute to state-dependent control of ven-
tilation and orexinergic neurons are located in the
hypothalamus, mainly in the lHyp and dHyp (Kuwaki
et al., 2008; Kuwaki, 2010; Li and Nattie, 2014; Vicente
et al., 2016). It was found in the present study that neu-
rons from these regions project to the cNTS and the LC
(Table 1). It is thus possible that some of the afferent pro-
jections from the hypothalamus to the cNTS and the LC,
observed in the current study, represent orexinergic neu-
rons that participate in state-dependent control of breath-
ing. This would also imply that this state-dependent
control might be well developed within the neonatal
period.
It is also known that the NTS plays a key role in the
consolidation of memories associated with object
recognition and aversive stimuli. The key finding is that
stimulation of the NTS with noradrenergic (Garcia-
Medina and Miranda, 2013), adrenergic agents (Clayton
and Williams, 2000) or glucocorticoids (Roozendaal
et al., 1999) facilitated the consolidation of aversive or
object recognition memories. It appears that activation
of the NTS works by increasing the release of nore-
pinephrine in the basolateral amygdala. There have been
numerous suggestions as to the anatomical neural path-
ways by which activation of NTS results in adrenergic
activation of the basolateral amygdala. There appear to
be direct activation pathways from the NTS to the amyg-
dala and at least some of the neurons projecting from the
NTS to the amygdala terminate on the basolateral amyg-
dala (Roozendaal et al., 1999; Clayton and Williams,
2000; Garcia-Medina and Miranda, 2013). It is also known
that LC neurons project to the amygdala and it has been
proposed that activation of the NTS may work through
activation of the LC (Clayton and Williams, 2000;
Garcia-Medina and Miranda, 2013). Finally, a more com-
plicated pathway from the NTS to the amygdala has been
proposed. In this pathway, activation of the NTS is pro-
posed to send projections to the Paragigantocellularis
nucleus, which activates the LC leading to release of
noradrenergic agents into the basolateral amygdala
(Clayton and Williams, 2000; Mello-Carpes and
Izquierdo, 2013).
Our present findings did not enable us to confirm the
full connectedness among the NTS, Paragiganto-
cellularis nucleus, the LC and the basolateral amygdala
that has been proposed from work in adults (Clayton
and Williams, 2000; Mello-Carpes and Izquierdo, 2013),
however we did find connections between the NTS and
the LC directly, as previously suggested (Clayton and
Williams, 2000; Garcia-Medina and Miranda, 2013). It
may be that this pathway can play an important part in
memory consolidation or it may represent a simpler path-
way used in young animals (neonates and juveniles) to
consolidate memory. Finally, we report here that
exposure of certain cNTS neurons to 15% CO
2
results
in activation of those neurons. It is likely that this stimulus,
which would undoubtedly be aversive, can activate the
cNTS and through whatever pathway, this could serve
to consolidate that aversive memory. In this line of rea-
soning, it may be that consolidation of aversive stimuli
may be present at a very early stage of development to
foster the survival of the organism whereas memories like
object recognition may not be consolidated until the
animal is considerably older (Nirogi et al., 2012).
Our speculation here will require further testing of the
development of memory consolidation and the
establishment of specific neuronal connections between
critical brain regions associated with memory
consolidation. We believe that our preparation as
described here would be most useful for such studies.
Anatomical analysis – considerations. The present
results on the anatomical connections between various
chemosensitive regions agree with a previous work
performed in adult rats; however, there is no study
performed in newborn rats to compare with our findings,
which makes our results unique. Nevertheless, it is
important to consider some points about our anatomical
analysis.
Some caution has to be taken when inferring afferent
projections from relatively small, highly localized areas.
For instance, the RTN involves a rather localized region
of chemosensitive neurons near the facial nucleus
(Mulkey et al., 2004). While in the present study some ret-
rograde labeling was observed near to the facial nucleus,
other data from Rosin et al. (2006) contradict our conclu-
sion that RTN neurons project to the LC, although these
authors found retrograde labeling from the SubC region,
concluding that RTN neurons do not project directly to
the LC. Another important point to be considered is that
contradictory results come from adult rats. It has been
suggested that ventilatory responses to CO
2
change dur-
ing neonatal development (Stunden et al., 2001; Putnam
et al., 2005; Davis et al., 2006). This raises the possibility
that the same could happen with brainstem interconnections.
L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 463

Thus, RTN neurons may project to the LC during
neonatal development but those projections could be lost
in adults.
A similar concern applies to the very small pre-Bo
¨tz
and Bo
¨tz regions where anterograde labeling studies in
adult rats did not indicate projections to the LC
(Ellenberger and Feldman, 1994). It could be that projec-
tions from these regions to the LC exist in neonates but
not in adults but such a conclusion would require further
study.
Electrophysiology
The question arises as to whether some of the afferent
connections between chemosensitive regions of the
brainstem directly carry chemosensitive information from
one area to another. This would most likely take the
form of intrinsically chemosensitive neurons from one
region projecting to another chemosensitive region. The
ventilatory response to hypercapnia is largely mediated
by CO
2
-sensitive neurons in the brainstem, referred to
as central chemosensitive neurons (Coates et al., 1993;
Nattie, 2001; Scheid et al., 2001; Putnam et al., 2004;
Ritucci et al., 2005). In the present study, to verify if a neu-
ron was chemosensitive we tested if the firing rate of the
neuron was altered by changes in CO
2
/H
+
(Figs. 9 and
10). Also, we verified if chemosensitive neurons were
intrinsically responsive to changes of CO
2
/H
+
by expos-
ing neurons to altered levels of CO
2
/H
+
in the presence
of synaptic block media (Figs. 9 and 10).
Interestingly, some neurons that project from the
cNTS to the LC and from the LC to the cNTS had
altered firing rate responses to hypercapnic acidosis
that persisted in the presence of SNB, suggesting that
these neurons are intrinsically responsive to changes in
CO
2
/H
+
. In older neonates (Conrad et al., 2009) and
adults (Nichols et al., 2009) 45–50% of the neurons from
the cNTS have firing rates that are increased by hyper-
capnic acidosis, while only 5–10% have firing rates that
are inhibited by hypercapnic acidosis. In neurons from
the LC of older neonates, between 20% and 40% of the
neurons have firing rates that are intrinsically increased
by hypercapnic acidosis (Nichols et al., 2008;
Gargaglioni et al., 2010) with no reported neurons that
are inhibited by hypercapnic acidosis. In our study, of five
cNTS neurons patched that project to the LC, four had fir-
ing rates increased by hypercapnia. These findings sug-
gest that a high percentage of cNTS neurons that have
afferent projections to the LC are intrinsically chemosensi-
tive. Although LC neurons are predominantly activated by
CO
2
(Filosa et al., 2002), in the present study we found
that two of the three LC neurons with afferent projections
to the cNTS had firing rates that were inhibited by hyper-
capnia. In the past, LC neurons that were inhibited by
hypercapnic acidosis were occasionally seen but the per-
centage was so low that such inhibited neurons have
never been reported (Putnam-unpublished data). It was
reported that epinephrine and norepinephrine both inhibit
the activity of cNTS neurons (Feldman and Moises,
1987), so inhibition of retrogradely loaded LC neurons
by hypercapnia may increase cNTS neuronal activity or
response to hypercapnia by reducing epinephrine and
norepinephrine release in cNTS from the terminals of
these LC neurons. Thus, these findings suggest that
highly specialized LC neurons are the ones with afferent
projections to the cNTS.
Based on these functional measurements, we assume
that upon exposure to hypercapnia cNTS neurons provide
excitatory input to the LC (+? in Fig. 11) while LC
neurons provide inhibitory input to the cNTS (?in
Fig. 11). These conclusions are based on the
assumption that both types of neurons that were studied
have an excitatory neurotransmitter phenotype. We also
do not currently know the relative contribution to central
chemosensitivity provided by different chemosensitive
regions. One way to consider relative contribution is by
the sensitivity to acid stimuli from different regions.
Using the same techniques in the same laboratory, we
know that the CI of chemosensitive neurons
(hypercapnic stimulus of 10% CO
2
) from the RTN of
neonatal rats (Ritucci et al., 2005) is larger (300%) than
the values of 150–175% reported for the CI of chemosen-
sitive neurons (hypercapnic stimulus of 15% CO
2
) from
the cNTS of neonatal rats (Conrad et al., 2009). Interest-
ingly, the chemosensitive response of LC neurons
appears to change with development. In LC neurons from
young neonates (<P10) the CI is about 235% but in LC
neurons from older neonates (>P10) it is considerably
less, around 125% (Gargaglioni et al., 2010). For neurons
from many other chemosensitive regions the CI lies
between the values of 125–300% (Putnam et al., 2004).
It will remain for future work to fully define how neurons
cNTS
dHyp / lHyp
PAG
LC
Kölliker-Fuse
Pre-Bötz/Bötz
RVL
RTN
SubC
A5
LC
dHyp / lHyp
cLC
SubC
PB
Kölliker-Fuse
A5
Pre-Bötz/Bötz/
RVL
MR
RTN
+?
-?
Fig. 11. Schematic diagram of the interconnections among several
putative chemosensitive regions of the brainstem, based on our
retrograde labeling data. Red arrows indicate afferent projections to
the LC while blue arrows represent afferent projections to the cNTS.
Thicker arrows indicate stronger retrograde labeling and thus more
projecting neurons while thinner arrows represent weaker labeling
and thus fewer projecting neurons. The blue arrow connecting LC to
cNTS is marked with a ‘‘?” to indicate that this connection may be
inhibitory (based on the electrophysiology data) but that this is
uncertain until the predominant neurotransmitter of these neurons is
known. The red arrow connecting cNTS to LC is marked with a ‘‘+?”
to indicate that this connection may be excitatory (based on the
electrophysiology data) but that this is uncertain until the predominant
neurotransmitter of these neurons is known. cNTS, caudal region of
the nucleus tractus solitarius near the area postrema; contralateral
LC, locus coeruleus on the side opposite the site of injection; dHyp/
lHyp, dorsomedial and lateral hypothalamus; LC, locus coeruleus;
MR, medullary raphe; PAG, periaqueductal gray; PB, parabrachial
nucleus; RTN, retrotrapezoid nucleus; SubC, subcoeruleus region;
Pre-Bo
¨tz/Bo
¨tz:, Pre-Bo
¨tzinger complex and Bo
¨tzinger complex; RVL,
rostroventrolateral reticular nucleus.
464 L. T. Lopes et al. / Neuroscience 324 (2016) 446–468

from various chemosensitive regions contribute to the
central chemosensitive network.
Nevertheless, it is clear that the anatomical
connections between the cNTS and the LC have the
potential to carry functional information about changes
in the levels of CO
2
/H
+
. This has recently been
confirmed for another part of the respiratory network,
where cNTS neurons that are responsive to a similar
level of acidosis as used in our study have been shown
to project to the ventral respiratory column (Huda et al.,
2012). Thus, the sharing of chemosensitive information
among various regions of the central chemosensitive net-
work may be widespread.
Summary – interconnections among chemosensitive
brain regions
Fig. 11 shows a schematic representation of the
interconnections among several putative chemosensitive
regions of the brain in neonatal rats, based on the
retrograde labeling data found in the present study. The
regions dHyp/lHyp, PAG, cLC, SubC, KF, A5, Pre-Bo
¨tz,
Bo
¨tz, RVL, cNTS, MR and RTN showed potential
projections to the cNTS (blue arrows) or to the LC (red
arrows). The thicker arrows indicate higher numbers of
neurons interconnecting the regions and the thin arrows
indicate fewer neurons making interconnections
between the regions (data based on Table 1). Most of
the neurons that project from cNTS to LC were
stimulated by hypercapnia Fig. 9), so it is assumed that
these projections are excitatory (+?), whereas some of
the neurons that project from LC to cNTS were inhibited
by hypercapnia (Fig. 10), suggesting that some of these
projections are inhibitory (?).
Significance and perspectives
Our findings clearly indicate that these anatomical
connections are present in rats 14–20 days after birth
and most likely at least as early as 7–10 days post birth.
The rhodamine bead injections were made in neonatal
rats aged P7–P10 and we presume that the projections
were intact at that age in order for retrograde labeling to
occur. We found that it required an additional 7–10 days
to see retrogradely labeled neurons with somas that are
clearly labeled. We assume that this time reflects the
long transport distance required in these neurons and
not additional time for projections to be established.
Further, at least for cNTS neurons projecting to the LC,
the pattern of sensitivity of firing rate to hypercapnia in
these neurons is unchanged in neurons from rats aged
P1 through the entire neonatal period (Conrad et al.,
2009) and into adulthood (Nichols et al., 2009). Based
on these findings, we suggest that both anatomical and
electrophysiological aspects of the respiratory network
are developed in neonatal rats as early as the first week
of life. It is possible that the network is established even
earlier but we were unable to perform retrograde labeling
studies in neonatal rats younger than P7 so it will be diffi-
cult to determine how early the network is established with
retrograde labeling techniques.
The data presented here (Figs. 9 and 10) as well as
those of others (Huda et al., 2012) suggest that there is
direct exchange of chemosensitive information between
central chemosensitive areas through the extensive
anatomical couplings that were observed here
(Fig. 11). Thus the alteration of firing rate by hypercap-
nic acidosis in chemosensitive neurons from one area
can directly result in altered firing rate in neurons from
other chemosensitive regions. These finding can help
explain an interesting phenomenon. It has long been
known that when multiple chemosensitive areas are
individually focally acidified, the sum of increased venti-
lation of all the individual areas can exceed the
increased ventilation induced by acidifying the whole
brain with increased inspired CO
2
(Coates et al.,
1993; Putnam et al., 2004). This would be explained
if focally acidifying one chemosensitive region of the
brainstem could activate other regions through excita-
tory projections. If this is occurring, it will also mean
that caution must be used when interpreting focal aci-
dosis experiments as indicating the contribution of just
the acidified region to the hypercapnic ventilatory
response. It is likely that such experiments could over-
estimate the contribution of a given area because acid-
ification of that area may result in stimulation of other,
non-acidified chemosensitive regions through the respi-
ratory network.
Finally, our work suggests several research
challenges for the future. The major challenge is to
describe the neurotransmitter phenotype of the afferent
projecting neurons between various chemosensitive
regions to determine the proportion of neurons that are
modulatory (e.g. positive for serotonin or orexin), that
are excitatory and that are inhibitory. Also, more
electrophysiological work needs to be done to determine
the extent to which intrinsically chemosensitive neurons
project between different chemosensitive regions.
Finally, these electrophysiological studies need to be
combined with immunohistochemical studies to
determine what neurotransmitters are released by the
projecting chemosensitive neurons in order to fully
describe exactly what information is being exchanged
between different chemosensitive regions in response to
hypercapnic acidosis.
As stated above, our work also suggests future
directions for the study of the role of various brainstem
regions in the development of memory consolidation
and whether differences may exist in the development
of aversive memories as opposed to other types of
memories.
CONCLUSIONS
The main conclusion from this study is that, in
neonatal rats, there are extensive anatomical
connections between different regions shown to be
involved in central chemosensitivity (and possibly
involved in the consolidation of memory). There are
no other studies in the literature about the respiratory
control network in neonates with which to compare
our results but our findings agree with other studies
L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 465

suggesting extensive inter-connections among many of
these regions in adult rats. Further we demonstrated
that at least some of these connections between
respiratory control areas involve the exchange of
chemosensitive information. Our work will contribute
to the understanding of the development of the
respiratory control network.
AUTHOR CONTRIBUTIONS
L.T.L., L.G.A.P., K.Y.L., A.N.I. and C.D.G. collected and
analyzed data. L.T.L., L.G.A.P., L.G. and R.W.P. wrote
and edited the manuscript.
CONFLICT OF INTEREST
There are no conflicts of interest.
Acknowledgments—This work was supported by American Heart
Association Great Rivers Affiliate Predoctoral Fellowship (ANI),
NIH R01-HL-56683 (RWP), Research Challenge Augmentation
Grant from Wright State University (RWP), Sa
˜o Paulo Research
Foundation (FAPESP), National Council for Scientific and
Technological Development (CNPq, Brazil) and a FAPESP
Fellowship Grants: 2008/57754-9 (to L.T.L.) and 2010/06210-9
(to L.G.A.P).
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