Catecholamine Inputs to Expiratory LaryngealMotoneurons in Rats

Wen-Jing Zhao,1 Qi-Jian Sun,2 Rui-Chen Guo,1 and Paul M. Pilowsky3*1Institute of Clinical Pharmacology, Qilu Hospital of Shandong University, Jinan, Shandong 250012, China2Australian School of Advanced Medicine, Macquarie University, Sydney, NSW 2109, Australia3Heart Research Institute and University of Sydney, Sydney, NSW 2042, Australia

ABSTRACTMany respiration-related interneurons and motoneurons

receive a catecholaminergic input, but the extent and

distribution of this input to recurrent laryngeal moto-

neurons that innervate intrinsic muscles of the larynx

are not clear. In the present study, we examined the

catecholaminergic input to expiratory laryngeal moto-

neurons in the caudal nucleus ambiguus by combining

intracellular labeling of single identified motoneurons,

with immunohistochemistry to reveal tyrosine hydroxy-

lase immunoreactive (catecholaminergic) terminal vari-

cosities. Close appositions were found between the two

structures, with 18 6 5 close appositions per moto-

neuron (n 5 7). Close appositions were more frequently

observed on distal rather than proximal dendrites. Axo-

somatic appositions were not seen. In order to deter-

mine the source of this input, microinjections of

cholera toxin B subunit (1%, 20 nl) were made into the

caudal nucleus ambiguus. Retrogradely labeled neurons,

located in the ipsilateral nucleus tractus solitarius and

the area postrema, were tyrosine hydroxylase-positive.

Our results not only demonstrate details of the extent

and distribution of potential catecholamine inputs to

the expiratory laryngeal motoneuron, but further indi-

cate that the inputs, at least in part, originate from the

dorsomedial medulla, providing a potential anatomical

basis for previously reported catecholaminergic effects

on the laryngeal adductor reflex. J. Comp. Neurol.

000:000–000, 2014.

VC 2014 Wiley Periodicals, Inc.

INDEXING TERMS: laryngeal motoneurons; tyrosine hydroxylase; nucleus tractus solitarius; intracellular recording;

respiration; cholera toxin B subunit; RRIDs AB: 10013220 and 477560

Catecholaminergic neurons comprise three subgroups

based on their complement of the catecholamine-

synthesizing enzymes, tyrosine hydroxylase (TH),

dopamine-beta-hydroxylase ("A1–7" cell groups) and

phenylethanolamine-N-methyltransferase ("C1–3" cell

groups) (Kalia et al., 1985). Catecholamine neurons

(TH-immunoreactive, TH-ir) are found in both the ventro-

lateral (A1/C1 cell groups) and the dorsomedial (A2/

C2 cell groups) medulla oblongata (H€okfelt et al., 1984;

Kalia et al., 1985; Pilowsky et al., 1990; Ellenberger

et al., 1990). In the ventrolateral medulla, TH-ir terminal

varicosities form close appositions with respiratory neu-

rons in the ventral respiratory column and motoneurons

in the rostral nucleus ambiguus (NA) (Sun et al., 1994).

Noradrenergic projections (TH-ir and dopamine-b-

hydroxylase-ir, but not phenylethanolamine-N-methyl-

transferase-ir) also arise from A5–7 cell groups and pro-

ject to the hypoglossal, facial, trigeminal motor nucleus

as well as motoneurons in the spinal cord (Card et al.,

1986; Grzanna et al., 1987; Lyons and Grzanna, 1988;

Aldes et al., 1990, 1992). Despite these extensive stud-

ies, it is still not clear if, and to what extent, recurrent

laryngeal motoneurons receive any TH inputs.

The caudal NA is mainly occupied by motoneurons

that project to, and regulate, intrinsic laryngeal muscles

via the recurrent laryngeal nerve (Bieger and Hopkins,

1987). Inspiratory laryngeal motoneurons innervate the

posteriod cricoarytenoid muscle, acting to open the

glottis during inspiration (Berkowitz et al., 1999). Expira-

tory laryngeal motoneurons (ELMs) innervate the thyro-

arytenoid, and other laryngeal constrictor muscles,

Grant sponsor: National Health and Medical Research Council ofAustralia; Grant number: 1024489; Grant sponsor: Australian ResearchCouncil; Grant numbers: DP110102110; G 11S 5957; Grant sponsor:National Nature Science of China; Grant number: 81202575.

*CORRESPONDENCE TO: Professor Paul M. Pilowsky, Heart ResearchInstitute, 7 Eliza St, Newtown 2042, Sydney, NSW, Australia.E-mail: paul.pilowsky@hri.org.au

Received October 21, 2013; Revised September 12, 2014;Accepted September 12, 2014.DOI 10.1002/cne.23677Published online Month 00, 2014 in Wiley Online Library(wileyonlinelibrary.com)VC 2014 Wiley Periodicals, Inc.

The Journal of Comparative Neurology | Research in Systems Neuroscience 00:00–00 (2014) 1

RESEARCH ARTICLE

acting to close the glottis and protect the airway from

aspiration during swallowing (Barillot et al., 1990; Yosh-

ida et al., 1998; Berkowitz et al., 2005; Ludlow, 2005).

The ELMs are partly controlled by monosynaptic con-

nections from respiratory neurons in the B€otzinger com-

plex and other ventral respiratory groups (Jiang and

Lipski, 1990; Ono et al., 2006; Sun et al., 2008). On

the other hand, during stimulation of the superior laryn-

geal nerve, ELMs are activated with a nonrespiratory burst

activity by neurons in the nucleus tractus solitarius (NTS)

around the obex level, which plays a critical role in coordi-

nating apnea, swallowing, and other airway protective

reflexes (Gestreau et al., 2000; Abu-Shaweesh et al.,

2001; Jean, 2001; Saito et al., 2002; Sun et al., 2011). In

addition, intravenous application of dopamine D1 receptor

antagonists is known to facilitate the laryngeal adductor

response in the rat (Feng et al., 2009). However, further

anatomical evidence of a dopamine input to the ELMs

remains unknown.

Given that TH-ir terminals and ELMs are both located

in the caudal NA (Kalia et al., 1985; Ludlow, 2005; Sun

et al., 2008), we hypothesized that there may be direct

contact between the two structures. This was examined

in the present study after intracellular labeling of ELMs

combined with immunohistochemistry to reveal TH-ir ter-

minals. In addition, in a separate tract-tracing study, our

findings not only confirm the existence of close apposi-

tions between the TH-ir varicosities and ELMs, but also

indicate that the TH inputs originate, at least in part,

from TH-ir neurons located in the dorsomedial medulla.

MATERIALS AND METHODS

Data were obtained from nine male Sprague–Dawley

rats (350–500 g, supplied by the Animal Resource

Centre, WA, Australia). All procedures were approved

by the Animal Care and Ethics Committee of Macquarie

University (http://www.nhmrc.gov.au/publications/

synopses/ea16syn.htm).

Animal preparationFive rats were anesthetized with sodium pentobarbi-

tone (72 mg/kg, ip, Virbac Animal Health, NSW, Aus-

tralia). The trachea, right femoral artery, and vein were

cannulated for artificial ventilation, blood pressure record-

ing and drug administration, respectively. Additional doses

of pentobarbitone sodium (3 mg/kg, iv) were adminis-

tered to maintain adequate anesthesia, as evidenced by a

stable blood pressure and phrenic nerve discharge, with

only minor autonomic responses (<10 mmHg increase in

blood pressure) in response to firm paw pinch. The left

phrenic and recurrent laryngeal nerves (RLN) were dis-

sected for recording or stimulation with standard bipolar

electrodes (Pilowsky et al., 1990). The rat was mounted

in a stereotaxic frame, paralyzed with pancuronium bro-

mide (1 mg/kg, with additional doses of 0.5 mg/kg, iv as

needed, AstraZeneca, Australia), and artificially ventilated

with oxygen-enriched air. Rectal temperature was main-

tained at 35–37�C with a homeothermic heating blanket

(Harvard Apparatus, Dover, MA). Part of the occipital and

parietal bones were removed to expose the dorsal sur-

face of medulla, and a laminectomy from C3 to C6 was

made to reduce brainstem movement.

Intracellular recording and labelingThe caudal NA was located by antidromic stimulation

of the RLN using low-impedance electrodes (5–10 Mx,

3M NaCl, Harvard Apparatus, SDR, Australia) to detect

field potentials. In three out of the five rats, when the

RLN recordings showed a weak respiratory activity the

left vagus was dissected for stimulation to locate the NA

and to identify ELMs (Gestreau et al., 2000). Intracellular

recordings were made with glass microelectrodes

(<1 lm tip diameter) containing 1.5% neurobiotin

(Vector Laboratories, Burlingame, CA) in 1M KCl (resist-

ance: 40–60 Mx). Only neurons with a stable membrane

potential more negative than 240 mV were labeled with

neurobiotin by using depolarizing current pulses (4–5 nA;

70% duty cycle for 3–5 minutes). After labeling, the elec-

trodes were rapidly removed from the cell and the

extracellular potential noted. One hour after labeling,

rats were perfused transcardially with 400 ml of saline,

followed by 400 ml of fixative containing 4% paraformal-

dehyde in 0.1M phosphate buffer (pH 7.4).

Microinjection of retrograde tracerA further four rats were anesthetized with urethane

(1.0–1.5 g/kg, ip) and maintained with additional doses

of pentobarbitone sodium (see above; 3 mg/kg, iv). The

right femoral vein was cannulated for drug administra-

tion. The left vagus nerve was dissected for stimulation

with standard bipolar electrodes. The rat was then

placed in a stereotaxic frame and a partial occipital cra-

niotomy performed to expose the dorsal surface of the

medulla oblongata. After the caudal NA was located by

antidromic stimulation of the vagus (10–15 mV, 0.2 ms),

cholera toxin B subunit (CTB; List Biological Laboratories,

Campbell, CA) was microinjected (1%, 20 nl) into the NA

at 0.5 mm caudal to the obex. After the CTB injection

(20–24 hours), the rat was perfused transcardially with

400 ml of saline, followed by 400 ml 4% paraformalde-

hyde in 0.1M phosphate buffer (pH 7.4).

ImmunohistochemistryThe medulla oblongata was removed from the rat

and post-fixed in the same fixative overnight. Vibrating

W.-J. Zhao et al.

2 The Journal of Comparative Neurology |Research in Systems Neuroscience

microtome sections (50 lm; Leica VT1200S) were

placed in 50% ethanol for 30 minutes. After three

washes with Tris phosphate-buffered saline (PBS) (Sun

et al., 1994), the sections from the intracellular labeling

experiments were placed in 10% normal horse serum

(Jackson ImmunoResearch, West Grove, PA) in Tris PBS

for 2 hours before incubation for 3 days in 10% normal

horse serum in Tris PBS (with 0.05% merthiolate) con-

taining ExtrAvidin-horseradish peroxidase (1:1,000 dilu-

tion, Sigma, St. Louis, MO, E2886) and the mouse

monoclonal antibody to TH (1:5,000; Sigma-Aldrich,

Cat. no. T1299, RRID: AB_477560) (Table 1). The mor-

phology of neurobiotin-filled neurons was revealed using

an imidazole-intensified diaminobenzidine reaction

(Pilowsky et al., 1991). Sections were then incubated

for 48 hours in a biotinylated donkey antimouse anti-

body (Jackson ImmunoResearch Laboratories, Lot no.

60651, 1:500 in 5% normal horse serum in Tris PBS)

and then incubated for 24 hours in ExtrAvidin-

horseradish peroxidase (1:1,500 dilution). TH-ir varicos-

ities were visualized with a nickel-intensified diamino-

benzidine reaction. Sections were mounted onto

gelatin-treated slides, dehydrated, and slides were cov-

erslipped with Ultramount No. 4 (Fronine, Australia).

Sections from the CTB microinjection experiments

were incubated for 48 hours in 10% normal horse

serum in Tris PBS (with 0.05% merthiolate) containing

goat anti-CTB (1:10,000 dilution; List Biological Labora-

tories, Campbell, CA, Cat. no. 703. RRID: AB

10013220. (Table 1), and mouse monoclonal antibody

to TH (1:2,000; Sigma-Aldrich, Cat. no. T1299, RRID:

AB_477560) (Table 1). After three washes in Tris PBS,

sections were incubated for 24 hours in 5% normal

horse serum in Tris PBS containing FITC-donkey anti-

sheep IgG (713-095-147, Jackson, 1:500 dilution) and

Cy3-donkey antimouse IgG (715-066-150, Jackson,

1:500 dilution) for visualization of CTB and TH-ir,

respectively. All incubations were performed at 4�C

with continuous gentle agitation and followed by 33

30-minute washes in Tris PBS. Sections were mounted

on microscope slides in Vectorshield mounting medium

(Vector Laboratories, Burlingame, CA).

Antibody characterizationThe suppliers specifications of the primary antibodies

used in this study are summarized in Table 1. Primary

antibodies were titrated on 4% paraformaldehyde-fixed

rat brainstem sections. The optimal concentrations of

the primary antibodies were determined as the dilution

at which the maximum number of immunoreactive

structures was observed with a minimal level of back-

ground staining.

The monoclonal anti-TH was derived from a hybrid-

oma produced by fusion of mouse myeloma cells and

splenocytes from an immunized mouse. Rat TH was

used as the immunogen. The antibody recognizes a sin-

gle band of �60 kDa on western blot from rat brain

(manufacturer’s technical information), and stained cat-

echolaminergic cells present in the brain and spinal

cord, and in sympathetic (Shi et al., 2008), chromaffin,

and entero-chromaffin systems. We confirmed that cell

morphology and regional distribution patterns of TH-

labeled neurons were identical to the results obtained

in previous studies (Pilowsky et al., 1990; Kawano

et al., 2006; Shi et al., 2008). A goat antiserum raised

against the CTB subunit (choleragenoid) was used. This

antibody recognizes a single band of CTB by western

blot (Xu et al., 2009). Goat antiserum to CTB did not

bind to any endogenous epitopes in the rat brain sec-

tions (Ge et al., 2010).

Biotinamide (neurobiotin) is an amino derivative of

biotin often used as an intracellular label for neurons.

Compared to biocytin and other neuronal labels, it is

more soluble, iontophoreses better, remains in cells lon-

ger, and can be fixed with formalin or glutaraldehyde.

Detection of neurobiotin using an avidin-peroxidase

technique revealed that only the one or two neuronal

cell somata that had been filled intracellularly were

unambiguously stained in any one brain.

Data analysisThe intracellularly labeled neurons were recon-

structed by light microscopy (340 objective) with a

camera lucida drawing tube. The criterion for a close

TABLE 1.

Primary Antibodies Used

Antigen Description of Immunogen

Source, Host Species, Cat.#,

Clone or Lot#, RRID Concentration/ Dilution

Tyrosine hydroxylase Rat tyrosine hydroxylase Sigma-Aldrich, mouse monoclonal,Cat#T1299, RRID: AB 477560

1 mg/ml, 1:5,000 (light),2.5 mg/ml, 1:2,000 (fluorescence)

Cholera Toxin Bsubunit (CTB)

B subunit (choleragenoid) List Biological Laboratories, goatantiserum, Cat#703, RRID: AB10013220

1:10,000

Catecholamine inputs to laryngeal motoneurons

The Journal of Comparative Neurology | Research in Systems Neuroscience 3

apposition was the presence of a side-by-side contact

formed between two structures with no discernible

intervening gap under an oil immersion objective lens

(3100). For estimation of the number and phenotype

of neurons in tract-tracing study, a one-in-four series of

50-lm coronal sections that contained the retrogradely

labeled neurons were chosen. All sections were exam-

ined and photographed using a Zeiss Axioimager Z2

microscope (Carl Zeiss Microimaging) with a Zeiss Axio-

cam HRm monochrome digital camera and Zeiss Imag-

ing software (AxioVision Rel. 4.8). Data are presented

as mean 6 SD.

RESULTS

Intracellular recording and labelingIntracellular recordings were made from nine ELMs

identified on the basis of their postinspiratory firing

pattern and antidromic responses following electrical

stimulation of either the RLN or vagus (Fig. 1A,B). They

were located within 1.5 mm caudal to the obex, 1.8–

2.2 mm lateral to midline, and 1.8–2.4 mm deep from

the dorsal surface of the medulla. The antidromic

response was confirmed using the collision test. Anti-

dromic latency was 2.5 6 0.6 ms (n 5 3) from the RLN,

or 0.8 6 0.1 ms (n 5 6) from the cervical vagus. Maxi-

mum membrane potential hyperpolarization was

256 6 12 mV, with an average membrane potential

swing of 11 6 3 mV (n 5 9) between inspiration and

expiration.

Seven of the nine labeled ELMs were reconstructed.

They were located �200–300 lm dorsal to TH-ir neu-

rons located in the A1 region of the ventrolateral

medulla (Fig. 1C,D). ELMs axons traveled dorsally for

variable distances before turning ventrolaterally to exit

the medulla. The other two ELMs were not

Figure 1. Identification of an expiratory laryngeal motoneuron (ELM). A: Intracellular recording from the ELM, showing its membrane poten-

tial in relation to phrenic nerve discharge (PND). B: Firing frequency (Freq) of the ELM and its antidromic response to stimulation (arrow)

of the recurrent laryngeal nerve (RLN). C: Micrograph of the intracellularly labeled ELM from (A). Note that the ELM (top arrow) is located

dorsomedially to TH-ir neurons clustered at the lower left corner of the micrograph (bottom arrow). L, lateral; V, ventral. D: Longitudinal

side-view of the medulla, showing schematically somata locations (asterisks) of the seven intracellularly labeled ELMs. The circled asterisk

indicates location of the identified ELM (A–C). VII, the facial nucleus; NAc, compact formation of the nucleus ambiguus. Scale bars 5 500

ms and 20 mV in A; 4 ms in B; 50 lm in C; 0.5 mm in D.

W.-J. Zhao et al.

4 The Journal of Comparative Neurology |Research in Systems Neuroscience

reconstructed because their intracellular injections

lasted less than 2 minutes and failed to stain the

recorded neurons sufficiently. In the ventrolateral

medulla, TH-ir varicosities were found around not only

TH-ir neurons in the C1/A1 area but also the NA

where ELMs were located. Close appositions, between

1 lm and 3 lm in diameter, were examined between

TH-ir varicosities and the seven ELMs under high

magnification (3100; Fig. 2). Most appositions were

found on distal dendrites (>100 lm from the soma)

rather than proximal dendrites (Fig. 3), and there was

no close apposition found on somata or axons of the

labeled ELMs. The reconstructed ELMs received 12 to

27 TH-ir close appositions with an average of 18 6 5

appositions per neuron (n 5 7).

CTB microinjectionMicroinjection of CTB (1%, 20 nl) was made into the

caudal NA in a further four rats. After immunohisto-

chemistry, the CTB-labeled neurons were found, apart

from the injection site, ipsilaterally in the dorsomedial

medulla, and contralaterally in the ventrolateral medulla

(Fig. 4). In the ipsilateral NTS there were many CTB-

labeled neurons that also expressed TH immunoreactiv-

ity (Fig. 5A–C). Interestingly, the double labeling was

only seen in small (somata with 10–15 lm in diameter),

rather than large (somata with 20–30 lm in diameter)

NTS neurons. Further counting, between 0.4 mm rostral

and 1.0 mm caudal to the obex, indicated that 58 6 22

out of 672 6 238 CTB labeled neurons were TH-

positive. In the ipsilateral area postrema, 48 6 20 out

of 130 6 19 CTB-labeled neurons, between 0.2 and

0.8 mm caudal to the obex, were found TH-positive. In

contrast, CTB labeled neurons in the ventrolateral

medulla, including both the ventral respiratory group

and the nucleus retroambiguus, were either inter-

mingled with or located above TH-ir neurons but were

not themselves TH-positive (Fig. 5D).

Figure 2. Close appositions between TH-ir varicosities and ELM. A: A high-power micrograph, showing one example of close appositions

between TH-ir varicosities (arrow) and an ELM’s dendrites (asterisks). B: Camera lucida reconstruction of an ELM (from 11 serial 50-lm

transverse sections). Close appositions are marked with black dots (not to scale). D, dorsal. Scale bars 5 10 lm in A; 50 lm in B.

Figure 3. Distribution histogram of close appositions, showing the

average number of TH-ir varicosities apposed closely onto the

ELM at increasing distances from the soma.

Catecholamine inputs to laryngeal motoneurons

The Journal of Comparative Neurology | Research in Systems Neuroscience 5

DISCUSSION

In this study, we demonstrate for the first time close

appositions between TH-ir varicosities and ELMs in the

rat. A subsequent tract-tracing study after CTB microin-

jection into the caudal NA revealed that retrogradely

labeled neurons were mainly present in the ipsilateral

NTS and area postrema, with a proportion of them colo-

calized with TH-immunoreactivity. These results suggest

that catecholamine projections may provide a direct

input to the ELMs in control of the laryngeal constrictor

muscles, laying a potential anatomical basis for previ-

ously reported dopamine effects on laryngeal adductor

reflex (Feng et al., 2009).

Catecholamine inputs to the ELMsIn the present study, we demonstrate that ELMs

receive close appositions from TH-ir varicosities. The

close appositions were found more commonly on the

distal rather than the proximal dendrites. No TH-ir vari-

cosity was observed to make close appositions with

somata or axons of the labeled ELMs. In comparison

with previous studies, TH-ir close appositions had a

similar distribution and density onto the respiratory

neurons in the B€otzinger complex and other ventral

respiratory neurons (Sun et al., 1994). In contrast,

fewer TH-ir varicosities were apposed closely to respira-

tory motoneurons in the rostral NA (Sun et al., 1994).

In addition, a similar pattern of TH inputs to motoneur-

ons in the hypoglossal nucleus has been reported, in

which TH-ir terminals were shown to form synaptic con-

nections with the hypoglossal motoneurons more fre-

quently on the dendrites rather than the somata (Aldes

et al., 1990). Based on our results that TH inputs to

ELMs are directed to their distal dendrites with a mod-

est number, we believe that the catecholamine inputs

may be more important in modulating responses to

other inputs, rather than in dominating, the firing of the

ELMs. Given the location on the distal dendrites, such

inputs may affect the electrotonic size of the neuron

differentially, so that distally located glutamatergic TH

inputs (all brainstem TH neurons are also glutamatergic)

Figure 4. Distribution of the retrogradely labeled CTB neurons in the medulla. A: Microinjection site of CTB (20 nl) into the caudal NA. CC,

central canal. B: Enlarged micrograph from boxed region B from (A), showing distribution of the CTB-labeled neurons in the dorsomedial

medulla. AP, area postrema; NTSi, ipsilateral nucleus tractus solitaries (NTS); NTSc, contralateral NTS. Please note that the CTB labeled

neurons are located in the ipsilateral, but not contralateral, NTS and AP. C: Enlarged micrograph from boxed region C from (A), showing

distribution of the CTB-labeled neurons in the ventrolateral medulla (circled by dashed line), with ventral surface indicated by the arrow.

Scale bars 5 500 lm in A; 100 lm in B,C.

W.-J. Zhao et al.

6 The Journal of Comparative Neurology |Research in Systems Neuroscience

are still effective (Minson et al., 1991; Pilowsky et al.,

1997; Stornetta et al., 2002). In the cortex there is a

differential amplification in both the amplitude, and

time, domain of the effect of glutamatergic inputs arriv-

ing on distal, compared with proximal, dendrites

(Branco and H€ausser, 2011). Additionally, a differential

distribution of sodium channels may ensure the fidelity

of dendritic transmission even when inputs are located

distally (Williams and Stuart, 2003).

We used a dual color diaminobenzidine reaction

(Pilowsky et al., 1991) to reveal the intracellular labeled

ELMs and TH-ir varicosities. In the present study, only

Figure 5. Images of dual-labeled CTB-ir and TH-ir neurons in the dorsomedial medulla, after the CTB microinjection into the caudal NA. A:

Low-power micrographs of the dorsomedial medulla, showing distributions of the CTB-ir neurons (green, A1), TH-ir neurons (magenta, A2),

and their merged image (white, A3). B: Enlarged micrographs of the boxed area B from (A), showing the CTB-ir neurons (B1), TH-ir neurons

(B2), and their merged images (arrows in B3) in the AP. C: Enlarged micrographs of the boxed area C from (A), showing the CTB-ir neurons

(C1), TH-ir neurons (C2), and their merged images (arrows in C3) in the NTS. D: Low-power micrographs of the ventral medulla, showing

the CTB-ir neurons (green, D1), TH-ir neurons (magenta, D2), and their merged image (white, D3) in the ventrolateral medulla of the con-

tralateral side. In the merged image, the mixture of CTB-ir and TH-ir neurons (boxed region) is enlarged at the upper left corner. Note that

the CTB-ir neurons are generally located more dorsally to the TH-ir neurons and no double-labeled neurons are observed. Scale bars 5 75

lm in rows A and D; 10 lm in rows B and C.

Catecholamine inputs to laryngeal motoneurons

The Journal of Comparative Neurology | Research in Systems Neuroscience 7

side-by-side contacts between reconstructed ELMs and

TH-ir varicosities were counted as close appositions.

While this criterion reduces the chance of false-positive

counts, it also inevitably underestimates the actual

number of close appositions on ELMs. On the other

hand, a close apposition is not definitive evidence of

the existence of synapse. Every close apposition cannot

be guaranteed to form a synaptic connection between

the two structures. In a previous study, we demon-

strated that about half of the close appositions

between substance-P-containing varicosities and sympa-

thetic preganglionic neurons formed synapses or direct

contacts when examined by electron microscopy (Pilow-

sky et al., 1992). Nevertheless, examination of close

appositions provides a useful and effective way to

assess the distribution of neurochemical inputs over

the entire somatodendritic tree of neurons and to com-

pare neurochemical inputs among different neuronal

groups (Paul et al., 1992; Murphy et al., 1995; Sun

et al., 2003). Given that our criteria will underestimate

synaptic input, but on the other hand some true synap-

ses may be missed or falsely classified, we believe that

our estimate may be reasonably accurate.

Origin of the catecholamine inputsAfter microinjection of CTB, we found that the retro-

gradely labeled neurons were located ipsilaterally in the

dorsomedial medulla and contralaterally in the ventro-

lateral medulla, including the nucleus retroambiguus

(Holstege, 1989). These results are in good agreement

with previous studies (Hayakawa et al., 2000; Ono

et al., 2006; Van Daele and Cassell, 2009). A novel

finding of this study, however, is that a proportion of

the CTB-labeled neurons in the NTS and area postrema

were also TH-ir-containing. In contrast, none of the

CTB-labeled neurons in the ventrolateral medulla were

TH-positive. The much closer proximity of unlabeled TH

neurons in the ventrolateral medulla to the injection

site argues against the findings being due to nonspe-

cific uptake by axons of passage. Similarly, the finding

of labeling in the contralateral, but not ipsilateral, NTS

also argues against the notion that the findings

occurred as a result of nonspecific uptake. However,

we cannot exclude that the injection also involves the

neurons surrounding the caudal NA, resulting in a small

portion of the retrogradely labeled neurons that do not

originate from the motoneurons but neurons of other

systems. Our study also agrees with those previous

studies that demonstrate monosynaptic inputs from

contralateral neurons of the ventral respiratory group to

recurrent laryngeal motoneurons (Ono et al., 2006;

Shiba et al., 2007). Although respiratory neurons them-

selves are not TH-positive (Ellenberger et al., 1990;

Pilowsky et al., 1990), we cannot rule out the possibility

that TH-ir neurons from other brainstem regions may

provide inputs to the ELMs, since a tracing time of less

than 24 hours in the present study may limit transport

to sites more rostral than the brainstem. The extensive

surgical preparation necessary to identify the injection

sites precluded recovery from anesthesia in these

experiments, which were all acute and terminal. For

example, noradrenergic neurons are known to project

from A5–7 cell groups to hypoglossal, facial, trigeminal

motor nucleus as well as motoneurons in the spinal

cord (Card et al., 1986; Grzanna et al., 1987; Lyons

and Grzanna, 1988; Aldes et al., 1992).

Functional implicationsIn the present study, we demonstrate that ELMs

receive an apparently modest number of close appositions

from TH-ir varicosities. The precise relationship of input

numbers to functional effects observed is unclear. Never-

theless, it is noteworthy that most of the neurotransmitter

products of TH neurons are metabotropic, so that at

times of extreme activity, when release from these sites

is likely to include catecholamine containing dense core

vesicles, the functional effects of even a small number of

inputs may be quite large. Effects of catecholamine inputs

to respiratory neurons are known to be different, depend-

ing on catecholamine subpopulations (Murakoshi et al.,

1985; Errchidi et al., 1990; Li et al., 2008). For example,

noradrenaline application to the brainstem has predomi-

nantly an inhibitory effect on respiratory neurons, whereas

dopamine application increased the respiratory frequency

(Murakoshi et al., 1985; Errchidi et al., 1990). During elec-

tromyographic recording from the thyroarytenoid muscle,

a blockade of dopamine D1 receptor was found to reduce

the response latency, but increased the response ampli-

tude of the laryngeal adductor response (Feng et al.,

2009). The thyroarytenoid is one of the main laryngeal

constrictor muscles that are controlled by the ELMs

(Ludlow, 2005). More functional studies are obviously

needed to clarify what exact role played by the TH inputs

to the ELMs.

CONFLICT OF INTEREST

We declare that there is no known or potential conflict

of interest including any financial, personal or other rela-

tionships with other people or organizations within 3 years

of beginning the submitted work that could inappropri-

ately influence, or be perceived to influence, our work.

ROLE OF AUTHORS

All authors had full access to all the data in the

study and take responsibility for the integrity of the

W.-J. Zhao et al.

8 The Journal of Comparative Neurology |Research in Systems Neuroscience

data and the accuracy of the data analysis. Study con-

cept and design: Paul M. Pilowsky, Qi-Jian Sun, Wen-

Jing Zhao, and Rui-Chen Guo. Acquisition of data: Wen-

Jing Zhao and Qi-Jian Sun. Analysis and interpretation of

data: Wen-Jing Zhao and Qi-Jian Sun. Drafting of the

article: Wen-Jing Zhao. Critical revision of the article for

important intellectual content: Paul M. Pilowsky. Statis-

tical analysis: Wen-Jing Zhao. Obtained funding: Paul M.

Pilowsky, Wen-Jing Zhao, and Qi-Jian Sun. Administra-

tive, technical, and material support: Qi-Jian Sun and

Paul M. Pilowsky. Study supervision: Paul M. Pilowsky.

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