Neuroscience 324 (2016) 446–468
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*

aDept of Animal Morphology and Physiology. São Paulo

State University, FCAV, Jaboticabal, SP, Brazil

bDept 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% CO2), even in synaptic

blockade medium (SNB) (high Mg2+/low Ca2+). 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.
http://dx.doi.org/10.1016/j.neuroscience.2016.03.036
0306-4522/� 2016 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; Bötz, Bötzinger 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, Kölliker-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-Bötz, Pre-
Bötzinger 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.

446
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. � 2016 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 CO2 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

CO2/H
+ with an increased firing rate (Elam et al., 1981;

http://dx.doi.org/10.1016/j.neuroscience.2016.03.036
mailto:robert.putnam@wright.edu
http://dx.doi.org/10.1016/j.neuroscience.2016.03.036


L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 447
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 CO2 (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 CO2

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% CO2; Putnam et al., 2004), the CI of

LC neurons from young neonates is fairly large 180–

220% (measured at 7.5%, 10% and 15% CO2; Putnam,

unpublished data) while it is very low in LC neurons from

older neonates (115–135% measured at 7.5%, 10% and

15% CO2; 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% CO2 (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% CO2; 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 CO2 (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 a adrenergic 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



448 L. T. Lopes et al. / Neuroscience 324 (2016) 446–468
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 lL or

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%

CO2 and rapidly decapitated. The brain was removed

and placed in 4–6 �C artificial cerebral spinal fluid

(aCSF) solution equilibrated in 95% O2/5% CO2 (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 �1 hr
at room temperature in aCSF equilibrated with 95%

O2/5% CO2 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 MX with a Narishige

Model PC-10 pipette puller. The bath was grounded with

an Ag–AgCl2 wire. Pipettes were filled with pipette filling

solution (see Solutions).



BA

C D

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
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 �1 GX seal was obtained.

For cNTS neurons, after the GX seal 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 GX seal 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% CO2 to the same perfusate bubbled with 15% CO2.

We chose to use this high level of hypercapnia (15%

CO2) 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% CO2 (Conrad et al., 2009). Similarly, 15% CO2

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% CO2) (Li and Putnam, 2013). Thus

the electrophysiology experiments as designed here allow

for the differentiation of whether a neuron is chemosensi-

tive or not.



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;

Bötz, Bötzinger 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
After the maximum effect of hypercapnia on firing rate

was reached, the perfusate bubbled with 15% CO2 was

changed back to 5% CO2, 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

MgSO4, 26 NaHCO3, 1.24 NaH2PO4, 10 glucose, and

2.4 CaCl2, equilibrated with 95% O2/5% CO2 gas, pH

�7.45. SNB was the same for aCSF except for (in mM):

121 NaCl, 11.4 MgSO4, and 0.2 CaCl2, plus

carbenoxolone (CAR; 100 lM) to block gap junctions.

Hypercapnic solutions were identical to aCSF and SNB

solutions except for equilibration with 85% O2/15% CO2

gas, pH �6.9–7.0. Paraformaldehyde solutions were



L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 451
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 Na2HPO4, and 1.47 NaH2PO4,

pH 7.4. The whole-cell pipette filling solution contained

(in mM): 130 K-gluconate, 0.4 EGTA, 1 MgCl2, 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
Table 1. Numbers of retrogradely labeled neurons in various brainstem region

Scoring: ‘‘0”: 0–1 neuron identified; ‘‘1”: 2–5 neurons identified. The numbers in black repre

represents a median labeling pattern. ‘‘3”: >10 neurons identified. The numbers in red re

involved in ventilatory control. N= 3; ND, not determined.
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% CO2 or

15% CO2, 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 P 6 0.05

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
s from rhodamine injections into either the LC or the cNTS

sents a weak labeling pattern. ‘‘2”: 6–10 neurons identified. The numbers in green

presents a strong labeling pattern. Highlighted areas in yellow represent regions



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 neuronswithin certain nuclei fromneonatal

rats injected with rhodamine beads into LC. The schematic drawings are taken from Paxinos and Watson (1997). (A) cNTS: caudal nucleus tractus

solitaries; 162 neuronswith rhodamine-loaded somawere counted in 12 slices between Bregma�13.80 and�14.40 mm; of these, 62%neuronswere

localized to the dorsolateral, commissural andmedial portion of the cNTS (red circle). (B)PAG:periaqueductal gray: 90 neuronswith 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 neuronswith rhodamine-loaded somawere counted in 14 slices betweenBregma�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-Bötz:

Pre-Bötzinger 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-Bötz Complex (red circle). (G) Bötz: Bötzinger complex; 27 neuronswith rhodamine-loaded somawere counted in six

slices between Bregma�11.96 and�12.48 mm; of these 70%were in the dorsal part of the Bötz Complex (red circle). (H) lHyp: lateral hypothalamus;

38 neurons with rhodamine-loaded somawere counted in 12 slices between Bregma�1.20 and�4.56 mm; of these 70%were in the dorsal part of the

Bötz 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



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
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), Kölliker-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-Bötzinger

and Bötzinger complex (Pre-Bötz/Bötz). 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).



L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 455
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-Bötz and Bötz region. In the Pre-

Bötz and Bötz 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
CC
Hy Hy

NTSNTS

DMNDMN

AP

Hy Hy
CC

NTSNTS

DMNDMN

AP

CC

Hy

NTS

DMN

A

CC

Hy

NTS

DMN

AP

Brightfield Brightfield andA

B

Fig. 5. Microinjection of rhodamine beads into the cNTS of neonatal rats.

brightfield and fluorescence imagens and the right column shows fluorescenc

general area of the hypoglossal (Hy) nucleus is marked, and the central cana

(DMN) and the NTS are delineated by white dotted lines. (A) NTS Hit. Note in

lie entirely within the NTS. (B) NTS Miss. Note in the fluorescence image tha

bar in each image represents 300 lm.
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-Bötz/Bötz

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
Hy

NTS

DMN

P

Hy

NTS

DMN

Hy Hy

NTSNTS

DMNDMN

AP

CC

CC
Hy Hy

NTSNTS

DMNDMN

AP

 Fluorescent Fluorescent

The left column shows brightfield images, the middle column shows

e images of a medullary slice containing the cNTS. For orientation, the

l (CC), the area postrema (AP), the dorsal motor nucleus of the vagus

the fluorescence image that the rhodamine beads (red fluorescence)

t the rhodamine beads lie entirely outside of the NTS. The calibration



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 neuronswith 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 neuronswith rhodamine-loaded somawere 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 neuronswith rhodamine-loaded somawere

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 somawere counted in 9 slices betweenBregma�10.44 and�10.80 mm; of these, 100%were localized to the caudal region and central area of

theRTN (red circle). (F) KF: Kölliker-Fuse; 41 neuronswith rhodamine-loaded somawere counted in six slices betweenBregma�8.72 and�8.80 mm;

of these, 83%were localized to the ventral portion of theKF (red circle). (G)Pre-Bötz: Pre-Bötzinger complex; 64 neuronswith 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-Bötz (red circle). (H)

Bötz: Bötzinger complex; 50 neuronswith rhodamine-loaded somawere counted in six slices betweenBregma�11.96 and�12.48 mm; of these, 72%

were localized to the ventral portion of the Bötz (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



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
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-Bötz and Bötz 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-Bötz and Bötz

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



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
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 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
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% CO2 vs. 1.26 ± 0.5 Hz

in 15% CO2, 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% CO2 vs.

0.27 Hz in 15% CO2, 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 CO2 chemosensitive.

In two of the three neurons, the addition of hypercapnia

resulted in a reversible decrease in firing rate (1.67 Hz

in 5% CO2 vs. 0.94 Hz in 15% CO2; 0.19 Hz in 5% CO2

vs. 0.01 Hz in 15% CO2, Fig. 10C). In the third, firing

rate was unchanged or slightly decreased by

hypercapnia (0.4 Hz in 5% CO2 vs. 0.33 Hz in 15%

CO2, 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.



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%CO2) in

the presence of aCSF. (A2) The integrated firing rate (action potentials per 10 s) in normocapnic (5% CO2) and hypercapnic (15% CO2) solutions.

(B1) The firing rate response of the same neuron as in A to hypercapnic acidotic solution (15% CO2) in the presence of synaptic block medium

(SNB). (B2) The integrated firing rate of the same neuron as in A in normocapnic (5% CO2) and hypercapnic (15% CO2) 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% CO2) in

the presence of aCSF. (A2) The integrated firing rate (action potentials per 10 s) in normocapnic (5% CO2) and hypercapnic (15% CO2) solutions.

(B1) The firing rate response of the same neuron as in A to hypercapnic acidotic solution (15% CO2) in the presence of synaptic block medium

(SNB). (B2) The integrated firing rate of the same neuron as in A in normocapnic (5% CO2) and hypercapnic (15% CO2) 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



462 L. T. Lopes et al. / Neuroscience 324 (2016) 446–468
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-Bötz/Bötz, 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 raphé

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). Raphé 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-Bötz/ Bötz, MR and RTN.

Afferent projections to the cNTS have previously been

observed from the ventral medullary respiratory group,

and specifically from the RVL, Bötz and Pre-Bötz (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



L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 463
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-Bötz 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 CO2 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% CO2 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 CO2 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.



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-Bötz/Bötz:, Pre-Bötzinger complex and Bötzinger complex; RVL,

rostroventrolateral reticular nucleus.

464 L. T. Lopes et al. / Neuroscience 324 (2016) 446–468
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-Bötz

and Bötz 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 CO2-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 CO2/H
+ (Figs. 9 and

10). Also, we verified if chemosensitive neurons were

intrinsically responsive to changes of CO2/H
+ by expos-

ing neurons to altered levels of CO2/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

CO2/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

CO2 (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% CO2) 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% CO2) 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



L. T. Lopes et al. / Neuroscience 324 (2016) 446–468 465
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 CO2/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-Bötz,

Bötz, 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 CO2 (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



466 L. T. Lopes et al. / Neuroscience 324 (2016) 446–468
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), São 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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