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Respiratory Physiology & Neurobiology 185 (2013) 20– 29

Contents lists available at SciVerse ScienceDirect

Respiratory Physiology & Neurobiology

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arotid chemoreceptor development in mice�

achiko Shirahata ∗, Eric W. Kostuk, Luis E. Pichardepartment of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA

r t i c l e i n f o

rticle history:ccepted 18 May 2012

eywords:reathingarotid sinus nerveene

a b s t r a c t

Mice are the most suitable species for understanding genetic aspects of postnatal developments of thecarotid body due to the availability of many inbred strains and knockout mice. Our study has shown thatthe carotid body grows differentially in different mouse strains, indicating the involvement of genes.However, the small size hampers investigating functional development of the carotid body. Hypoxicand/or hyperoxic ventilatory responses have been investigated in newborn mice, but these responsesare indirect assessment of the carotid body function. Therefore, we need to develop techniques of mea-

ypoxianbredutbred

suring carotid chemoreceptor neural activity from young mice. Many studies have taken advantage ofthe knockout mice to understand chemoreceptor function of the carotid body, but they are not alwayssuitable for addressing postnatal development of the carotid body due to lethality during perinatal peri-ods. Various inbred strains with well-designed experiments will provide useful information regardinggenetic mechanisms of the postnatal carotid chemoreceptor development. Also, targeted gene deletionis a critical approach.

. Why mice?

The critical role of the carotid body in the regulation of breathingas been recently emphasized in both animal and human studies.

n contrast to the traditional view, the input from the carotid bodys essential for proper function of the central chemoreceptors indult humans and animals (Timmers et al., 2003; Dahan et al., 2007;lain et al., 2009, 2010). However, the function and morphologyf the carotid body are not the same throughout life. Embryonicevelopment of the mammalian carotid body has been investi-ated in many species including humans (Boyd, 1937; Hervonennd Korkala, 1972; Smith et al., 1993), calves (Smith, 1924), sheepBlanco et al., 1984), cats (Clarke and Daly, 1985), rabbits (Kariyat al., 1990), rats (Smith, 1924; Rogers, 1965; Kondo, 1975) andice (Kameda et al., 2002). Further, studies of postnatal develop-ent of the carotid body include humans (Dinsdale et al., 1977),

heep (Blanco et al., 1984, 1988), pigs (Mulligan, 1991), cats (Carrollt al., 1993; Carroll and Fitzgerald, 1993), rabbits (Bolle et al., 2000;

� This paper is part of a special issue entitled “Development of the Carotid Body”,uest-edited by John L. Carroll, David F. Donnelly and Aida Bairam.∗ Corresponding author at: Department of Environmental Health Sciences, Johnsopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, MD1205, USA. Tel.: +1 410 614 5447; fax: +1 410 955 0299.

E-mail address: mshiraha@jhsph.edu (M. Shirahata).

569-9048/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.resp.2012.05.017

© 2012 Elsevier B.V. All rights reserved.

a limited number of references to point out that a variety of ani-mals have been examined. These studies have shown that majorchanges in the function of the carotid body occur at postnatal peri-ods, and the morphology of the carotid body continually developsafter birth.

The fetal carotid body responds to severe hypoxia, but carotidbody function is reset after birth (for reviews, Donnelly, 2000;Carroll, 2003; Gauda et al., 2009). Function of the fetal carotidbody was studied in sheep which were close to full term (Blancoet al., 1984). Functional changes of the carotid body during transi-tion from fetus to newborn were also studied in the sheep (Blancoet al., 1984, 1988). However, the developmental stages of fetusesand newborns are extremely variable among species (Evans andSack, 1973; Sterba, 1995). The sheep is born in a matured form;i.e., a newborn sheep is covered with a developed hair coat, andis able to see and walk. On the other hand, a rodent pup is bornunderdeveloped with no hair, closed eyes and limited motor skills.Thus, special care must be taken when dealing with developmentalstages for extrapolating data from one species to other species.

Although many animal species have been used as “modelspecies” in neurodevelopmental research, a recent trend showsthe dominant use of rats and mice (Clancy et al., 2007). Clearly,mice are the most suitable species for genetic studies because ofthe availability of many inbred strains and thousands of knockoutmice (Collins et al., 2007). A major disadvantage of the use of mice in

M. Shirahata et al. / Respiratory Physiology & Neurobiology 185 (2013) 20– 29 21

Fig. 1. Carotid body chemoreceptor neural activity from three inbred strains of mice (DBA/2J, A/J and FVB strains). Top: Raw activities of carotid sinus nerve in response tohypoxic challenges (gray bars, FIO2 0.15 and 0.1 for 90 s). Bottom: Mean chemoreceptor activity over time. The signal was processed via Fast Fourier Transformation andt r of aT three

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he Hann weighing function. It was normalized against the sum of the total powehe response of carotid body chemoreceptor activity to hypoxia varied among the

hallenge). PaO2 reached ∼40 mmHg when mice were ventilated with 10% O2 for 9

evelopment of the mouse carotid body has been mainly estimatedy measuring hypoxic or hyperoxic ventilatory response, which isn indirect assessment of the carotid body function. Why then aree bothering to use developing mice? Generally speaking, organevelopment of the mouse and the rat is similar (Schneider andorton, 1979). Can we just use the data obtained from the rat,articularly in carotid sinus nerve activity? The authors’ currentosition is against this idea based on the data of various inbredtrains of mice and the comparative studies between mice and rats.asal respiration as well as hypoxic and hypercapnic ventilatoryesponses are age and strain dependent (Balbir et al., 2008; Balbir,008; Arata et al., 2010). Further, VE (minute ventilation) adjustedo body size increases in mice from newborn to adult, but decreasesn rats (Mortola and Noworaj, 1985). In a comparative study Mor-ola showed that breathing frequency and tidal volume in newborn

ice (3 days old) increased in response to hypoxia (10% O2 for0 min), but in rats (2 days old) breathing frequency increased andidal volume decreased (Mortola et al., 1989). Carotid chemorecep-or neural responses to hypoxia in adult mice differ among severaltrains (Fig. 1). These data indicate that mice are not little rats, andhat we need to investigate each species and each strain separately.nce our investigation progresses, we shall obtain a more com-rehensive view as to the similarities and differences between thepecies and strains. Then, we can evaluate each animal model whennd how the model simulates human conditions. Here, we are sum-arizing the past findings related to postnatal development of theouse carotid body, and further suggest future directions of the

esearch.

. Carotid chemoreceptor neural output

Carotid chemoreceptor neural activity in mice has beenecorded mainly using an in vitro preparation from adultsDonnelly and Rigual, 2000; Kline et al., 2002; He et al., 2002;ong et al., 2003; Prieto-Lloret et al., 2007; Trapp et al., 2008;eng et al., 2010). Most of these studies have compared the neuralesponses to hypoxia between wild type mice and knockout mice.

e have observed that carotid chemoreceptor neural responsesn vivo differ in three inbred strains of mice (Fig. 1). In these stud-es, mice were ventilated with 100% oxygen maintaining PaCO2

al activity increased in DBA/2J mice, but barely changed in A/Jice. In FVB mice, the speed of the increase in neural activityas very fast, and activity was not sustained throughout the 10%

ll experiments. N2 challenge (30 s) was used to confirm the viability of the nerve.inbred strains. The carotid body of A/J mice responded only to severe stimulus (N2

O2 challenge. With milder hypoxia (15% oxygen) the neural out-put continually increased throughout the 90 s of the challenge inDBA/2J and FVB mice. Again, FVB mice showed a sharp rise in neu-ral output, and A/J mice did not show a significant increase. Carotidbody responses to anoxic stimuli were seen in all mice, but theresponse in A/J mice was delayed and much smaller. Because thesemice are raised in the same room in our facility where environ-ments are well controlled, the differences in neural responses tohypoxia most likely reflect genetic differences. Comparative studiesin these strains will provide important information as to the genera-tion of chemoreceptor neural activity and possibly hypoxic sensingmechanisms. Currently, developmental studies in carotid chemore-ceptor neural activity are lacking in mice. In rats, the carotid bodychemoreceptor neural response to hypoxia increases with postna-tal age. The response appeared to be negligible until ∼1 week old(Kholwadwala and Donnelly, 1992), and reached a plateau betweenone and two weeks after birth (Kholwadwala and Donnelly, 1992;Bamford et al., 1999). It requires future investigation to confirmthat this is also the case in mice. Due to its small size, recordingof carotid sinus nerve activity in newborn mice would be a greatchallenge, but in vitro techniques could be developed for older mice(e.g. >P7).

3. Measurement of breathing parameters

Several strains of mice have been used to examine their charac-teristics of breathing. We have summarized breathing parametersobtained from four inbred strains (C57BL/6, BALB/c, DBA/2J, A/J),two hybrids (C57BL/6–129SvEv, C57BL/10–129SvEv), offspring (F1)of two inbred strains (C57BL/6 × BALB/c), and some outbred stocks(Swiss-IOPS, ICR, Swiss CD1, Swiss) (Table 1). The data wereobtained from several publications. The timing of measurementsvaries among the studies. Renolleau et al. (2001a) have showndetailed time dependent changes in baseline breathing as well asventilatory responses to short-term (90 s) hypoxia and hypercap-nia between 1 and 48 h after birth. Their data indicate that basalbreathing patterns significantly change during the first 24 h afterbirth. Respiratory frequency and tidal volume increased with timeduring the initial 12 h after birth, and then decreased with time forthe next 12 h (24 h after birth). At ages less than P1 many investiga-

22 M. Shirahata et al. / Respiratory Physiology & Neurobiology 185 (2013) 20– 29

Table 1Breathing characteristic during first 24 h of life.

Strain Age (h) Methods Chamber temperature Frequency (bpm) TTOT (ms) Tidal volume (�L/g) References

Swiss-IOPSb 1 WBP 32 ◦C 115c 524c 3.0c Renolleau et al. (2001a)6 132c 454c 4.1c

12 160c 376c 5.5c

24 119c 503c 4.9c

Swiss CD1 P0a HO 35–36 ◦C 55 ± 7 1091c 12.7 ± 1.3 Robinson et al. (2000)

Swiss 3 WBP 30.5 ◦C 159c 377c 2.1c Dauger et al. (2001)12 144c 417c 4.6c

ICR P0a WBP NM 88.6 ± 21.0 677c 39.2 ± 9.4d Arata et al. (2010)e

BALB/c P0a 69.5 ± 13.9 863c 26.5 ± 8.0d

F1 of BALB/c and C57BL/6 P0a 142.8 ± 45.3 420c 15.1 ± 4.6d

C57BL/6 P0a 101.5 ± 26.6 591c 20.5 ± 6.3d

C57BL/6 6–12 HO 32–34 ◦C 120.1 ± 9.5 500c NM Erickson et al. (2001)C57BL/6–129SvEv 1 WBP 28–30 ◦C 95c 630 ± 54 3.1 ± 0.4 Renolleau et al. (2001b)e

C57BL/10–129SvEv 10–12 WBP 32 ◦C 127c 474c 5.3c Aizenfisz et al. (2002)

Data show mean values and in some cases ±SE.a Exact age (h) was not provided.b The original include two sets of delivery groups (vaginal and cesarean section), but only data from vaginal delivery group are presented here. WBP: non-restrained whole

body plethysmography; HO: head out plethysmography.

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c These values are estimated from the figures or numbers they presented.d These valued were not normalized by weight; NM: not mentioned.e The pups were delivered by cesarean section.

how tidal volume or minute ventilation here, because these valuesn small animals are semiquantitative in nature (Enhorning et al.,998; Gaultier and Gallego, 2008) and extremely varied among theeports.

Before we further discuss the ventilatory responses to hypoxia,e will address possible reasons of the variability among the stud-

es seen in Table 1 and Fig. 2. These include postnatal time, strains

ig. 2. Changes in breathing frequency with postnatal maturation (from P1). Substantial cs also observed among different strains. Measuring conditions may have influenced the resody plethysmography (Renolleau et al., 2001a); (�) Swiss CD1, head out plethysmograpDauger et al., 2001); (�) ICR, unrestrained whole body plethysmography (Arata et al., 2011 of BALB/c and C57BL/6, unrestrained whole body plethysmography (Arata et al., 2010

C57BL/6, head out plethysmography (Bissonnette and Knopp, 2001); (�) C57BL/6, unreslethysmography (Kazemian et al., 2001); (♦) DBA/2J, unrestrained whole body plethysm008).

and Gaultier’s group, who has performed extensive investigationin ventilatory control of young mice, has used Swiss outbred stocksas well as the C57BL–120SvEv hybrid. An inbred strain is producedby sister–brother mating or parent–offspring mating for at least20 consecutive generations. Therefore, except for the sex differ-ence, mice of an inbred strain are as genetically alike as possibleand are homozygous at virtually all of their loci. On the other hand,

hanges in breathing frequency occur initial several days after birth. A large variationults as well. Symbols and references as follows: (�) Swiss-IOPS, unrestrained wholehy (Robinson et al., 2000); (�) Swiss, unrestrained whole body plethysmography0); (�) BALB/c, unrestrained whole body plethysmography (Arata et al., 2010); (�)

); (�) C57BL/6, unrestrained whole body plethysmography (Arata et al., 2010); (trained whole body plethysmography (Dauger et al., 2004); (×) C57BL/6, head outography (Balbir, 2008); (©) A/J, unrestrained whole body plethysmography (Balbir,

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ackground is poorly characterized (Chia et al., 2005; Festing,010). Because of its homogenous and controlled genotype, an

nbred strain can characterize the environmental effects betterhan an outbred stock (Festing, 2010). Arata et al. (2010) specifi-ally addressed the strain differences in the control of breathingn their preliminary study, indicating a large variation of breathingarameters among different strains. Unfortunately, their data doot provide detailed recording conditions. Our data at P1 also showhe difference between the DBA/2J and A/J inbred strains (Fig. 2)Balbir, 2008). More detailed studies are necessary for the earlyhase of respiratory changes in other inbred strains of mice suchs C57BL/6 inbred mice.

Ventilatory responses to hypoxia depend on not only carotidody function but also the central respiratory control sys-em. Sleep–wake states influence the central respiratory controlystem: the ventilatory response to hypoxia is attenuateduring sleep compared to awake (reviewed by Shea, 1996;rimsky and Leiter, 2005). Therefore, the ventilatory responses

o hypoxia must be measured under a known sleep–wake state.leep–wake states can be classified by assessing the electroen-ephalogram (EEG), electromyogram (EMG) and eye movementelectrooculogram; EOG) (Brain Facts, Society for Neuroscience;ttp://www.sfn.org/skins/main/pdf/brainfacts/2008/sleep.pdf). Inhe adult mouse, EOG has been omitted due to the apparent tech-ical difficulties, and EEG and EMG have been used (Schaub et al.,998; Tagaito et al., 2001). However, EEG and EMG instrumenta-ion in neonatal mice is challenging, and it could induce stresseshich alter their behavior and ventilation. Karlsson et al. estab-

ished a two-state model (sleep and wake) for neonatal rats usingnly EMG and behavioral criteria (Karlsson and Blumberg, 2002;arlsson et al., 2004). Durand et al. (2005) also used EMG andehavioral indices to assess wake-sleep states in 5-day-old mice.e examined whether we can determine sleep–wake states using

xclusively behavioral indices in 7-day-old DBA/2J and A/J inbredice (Balbir et al., 2008). We found that coordinated movements

CM; defined as the prolonged movement of multiple limbs andhe head) were useful indices. They were observed only duringeriods of high EMG activity. The duration of CMs was not sig-ificantly different from the duration of high EMG in both strainsf mice. Further, the data indicate that the instrumentation of theMG appears to cause stress in neonatal mice. Accordingly, a non-nvasive method for sleep–wake assessment by monitoring theehavioral indices would be a more suitable approach to mea-ure ventilatory responses to gas challenges in 7-day-old mice.his approach has also been used in younger mice (up to 48 hfter birth) while measuring ventilation (Dauger et al., 2001): Sleepas defined as immobility in the recumbent position, and arousalas characterized by sudden neck and forepaw extension. In adult57BL/6J mice, more than 40 s of continuous inactivity has beenonfirmed as sleep (Pack et al., 2007). Currently, we need to fillhe gap to establish methods for adequately assessing sleep–waketates in mice at the ages between 7-day-old and adult.

Regarding sex, many reports shown in Table 1 and Fig. 2 pooledhe data from both of females and males or even did not mentionedex. Although sex differences were reported in hypoxic and hyper-apnic ventilatory responses in rats (Holley et al., 2012), Renolleaut al. (2001b) analyzed the sex effects and did not find any effectsn breathing parameters in the C57BL/6–129SvEv hybrid strain.

In terms of measuring techniques, Gautier and Gallegoddressed the problems and difficulties of measuring ventilationrom neonatal mice and recommended the use of unrestrainedhole body plethysmography (Gaultier et al., 2006; Gaultier and

he instrumentation of the EMG electrodes differently, the former

& Neurobiology 185 (2013) 20– 29 23

aggressively and the latter submissively (Balbir et al., 2008). In thisstudy the instrumentation of EMG electrode lightly restrained themovement of animals and possibly caused discomfort. Thus, therestraint due to head out plethysmography likely evokes differ-ent responses in different strains. A comparison between severalmouse strains must be performed carefully in studies using thistechnique. Most investigators are aware of the importance oftemperature control, and therefore, the recording chamber waswarmed to thermoneutral ranges. Gaultier et al. (2006) assumedthe thermoneutral zone for mouse pups to be 32–33 ◦C that isequivalent to the temperature inside the litter. One early study withhead out plethysmography used room temperature (26 ◦C) andobtained an extremely slow breathing frequency (37 ± 15 bpm) atP0 (Burton et al., 1997). This observation is not included in Table 1,but gives interesting insights regarding the effects of temperatureon breathing.

4. Development of the hypoxic ventilatory response

As described above, breathing measurement in newborn micerequires the careful control of many factors. Therefore, to discussthe hypoxic ventilatory response we have selected data in whichmeasurement conditions were clearly described and they weremaintained in optimal conditions. Fig. 3 shows the hypoxic ven-tilatory response during the early phase of life (<24 h). Most datawere obtained from Gallego and Gautier’s laboratory (Dauger et al.,2001; Renolleau et al., 2001a,b; Aizenfisz et al., 2002). In two exper-iments (Swiss-IOPS and C57BL/6–129SvEv) the hypoxic challengewas given using a gas mixture of 10% O2/3% CO2/87%N2 for 90 s. Wecan observe a clear difference in TTOT (total breathing cycle time)at 1 h of age between the two experiments. The reason for this dif-ference is not apparent, but genetic differences (strain differences)may be a possible explanation. A striking observation here is thepresence of the hypoxic ventilatory response at very early age (1 h)due to mostly increased VT (tidal volume). 3% CO2 added to theinspiratory gas may contribute to the increase in ventilation, but at3 h of age, severe hypoxia (5% O2) without CO2 clearly increased VTand decreased TTOT resulting increased VE. These data suggest thatmice show the hypoxic ventilatory response at a very early age.Within Swiss IOPS (Renolleau et al., 2001a), the hypoxic ventila-tory response is weaker at 1 and 6 h of age compared to 12 h of age.The response did not significantly change among 12, 24 and 48 hof age. Two studies examined the later phase of postnatal devel-opment of the hypoxic ventilatory response (Robinson et al., 2000;Bissonnette and Knopp, 2001). Both used head out plethysmogra-phy. When we focus on the early time periods in hypoxia, relativechanges in ventilation decrease from P1 and the response plateauedat ∼P20 in both Swiss CD1 outbred and C57BL/6 inbred mice. Insummary, available data indicate that the ventilatory response tohypoxia is present even 1 h after birth. The responses of frequencyand tidal volume to hypoxia develop differently with age, but theyappear to reach steady state level around 3 weeks of age.

An important consideration is whether we can use breathingparameters from newborn mice as an indication of carotid bodyfunction. Since we do not have any information about carotid bodyneural output of newborn mice, here we examined the data fromrats, hoping that new insights may be obtained in the relationshipbetween carotid body function and hypoxic ventilatory responseduring postnatal development. Liu et al. (2006) have found dynamicchanges in hypoxic ventilatory responses in rats from P0 to P21.Increases in breathing parameters in response to early time peri-

24 M. Shirahata et al. / Respiratory Physiology & Neurobiology 185 (2013) 20– 29

Fig. 3. Hypoxic ventilatory response during 24 h of age. Values within initial 1 minof hypoxic challenge were taken from figures and tables of several references(listed below). All experiments were performed using unrestrained whole bodyplethysmography except for the last bar (P0) which was used head out plethys-mography. Gas contents during challenge varied: some are pure hypoxia (5% or7.4%, indicated in the top panel) and others are 10% O2/3% CO2/87% N2 (*). Data aremean ± SE. Swiss derived outbred stocks were mainly used for the study: Swiss-IOPS(2e

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Fig. 4. Age-dependent changes in the volume of the carotid body in the DBA/2J andA/J strains. Carotid bodies were obtained at 1 day (1D), 1 week (1W), 2 weeks (2W),and 4 weeks (4W) of age. Scattergram shows the distribution of the volume of thecarotid body in the DBA/2J (�) and A/J (©) mouse strains. Mean of the values isrepresented by the black line through the plotted values. No significant difference isfound between the strains in the growth of the carotid body at 1D and 1W. DBA/2Jstrain has a significant increase in the volume of the carotid body from 1W to 2W,and has a significantly greater carotid body volume than the A/J strain at 2W and4W. A/J strain shows no significant growth from 1D to 4W. Statistical analysis wasperformed using one-way ANOVA (between 4 age groups within a strain) and aStudent’s t-test (between the strains at a corresponding age group). *P < 0.05, values

§

Renolleau et al., 2001a), Swiss (Dauger et al., 2001), Swiss CD1 (Robinson et al.,000), C57BL/6–120SvEv (Renolleau et al., 2001b), 129SvEv–C57BL/10 (Aizenfiszt al., 2002).

t P13, but subsequently the response recovered and graduallyeached a steady-state toward P21 with a predominant increase inidal volume. Usually, the hypoxic ventilatory response during earlyime periods of the hypoxic challenge is considered to be mediatedy the carotid body. However, carotid body chemoreceptor neu-al activity minimally responded to anoxic stimuli until ∼P7 andfter P10 the response became significantly larger than the youngerges (Kholwadwala and Donnelly, 1992). A similar response pat-ern was seen in the calcium response of chemoreceptor glomus

significantly different between strains. P < 0.05, values significantly different from1D. #P < 0.05, values significantly different from 1W.

Reproduced from (Kostuk et al., 2012).

VE response to hypoxia between P6 and P8 was almost comparableto that of P19 and P21. In mice, the hypoxic ventilatory responseis seen earlier than in rats. The dichotomy between the breath-ing response and the carotid body response to hypoxia at earlypostnatal ages in the rat suggests that the breathing responses arenot appropriate to be used as a surrogate of carotid body func-tion in neonatal rodents. It is possible that the hypoxic ventilatoryresponse in these animals is mediated by chemosensitive organsother than the carotid body such as the pulmonary neuroepithelialbody or adrenal chromaffin cells (Nurse et al., 2006). To confirmthese points, more comprehensive and comparative studies arerequired in measuring carotid body neural output and breathingin response to similar stimuli at the same developmental stages.

5. Morphological development of the carotid body

Morphology of the carotid body also matures postnatally. It hasbeen shown that the innervation of glomus cells is not fully maturedat birth in the rabbit (Kariya et al., 1990; Bolle et al., 2000) and therat (Kondo, 1975). The volume of the carotid body continues torise from birth to 10 years old in humans (Dinsdale et al., 1977;Heath and Smith, 1992). Our study in DBA/2J and A/J inbred strainshas shown that the growth of the carotid body is age- and straindependent (Kostuk et al., 2012). Carotid bodies and glomus cellsat 1-day and 1-week-old mice demonstrated very similar grossmorphology in both strains. At 2 weeks of age, the carotid bodyof DBA/2J strain became clearly larger compared with the carotidbodies of younger ages. In A/J mice, the size of the carotid body didnot apparently change through 1 day to 4 weeks old, but the shapeof the carotid body became irregular at 2 weeks old. At 4 weeks old,the carotid body of A/J strain began to exhibit a phenotype similarto that reported in the adult A/J’s carotid body (Yamaguchi et al.,2003). Volumetric measurements show (Fig. 4) that the carotid bod-ies of both strains were similar at 1 day old. There was a trend ofsmall increases in the carotid body size from 1 day to 1 week ofage in both strains. In DBA/2J mice, the carotid bodies grew rapidly

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stimated total glomus cell volume at 1 day was equivalent in bothtrains. In the DBA/2J mice, it increased up to 2 weeks of age and wasaintained till 4 weeks, but in the A/J mice, it reached maximum

t 1 week, and then, decreased continually (Kostuk et al., 2012).e have recently measured the carotid body volume of C57BL/6ice at 2 weeks of age. The mean value (4.3 ± 0.3 × 106 �m3, n = 5)as a midpoint between the carotid bodies of DBA/2J and A/J mice.

urther studies at different ages will confirm whether the carotidody of the C57BL/6 strain develops slowly compared to that of theBA/2J strain, or if the carotid body of the C57BL/6 does not reach

he volume of that of the DBA/2J strain. Taken together, these datahow the differential postnatal growth of the carotid body in differ-nt inbred strains and indicate that genetic factors are regulatinghe development of the carotid body. This explains the large vari-bility of the carotid body volume in humans (Dinsdale et al., 1977;eath and Smith, 1992) and cats (Clarke and Daly, 1985), because

he genetic background in humans and cats is heterogeneous.Although the size of the carotid body may not be completely

oupled with the function of the carotid body, some data indicatessociation between the anatomy and function of the carotid body.or example, the exposure to hyperoxia during early developmen-al periods induces atrophy of the carotid body and decreases theumber of glomus cells in rats. In these animals, the carotid sinuserve response to hypoxia is extremely attenuated (Bisgard et al.,003; Prieto-Lloret et al., 2004). Further, it has been shown thathe synaptic nature continues to develop up to 5 weeks in the ratKondo, 1976) and the rabbits (Bolle et al., 2000). These studiesndicate that afferent synapses continue to increase during thiseriod. Certainly, these ultrastructual changes would contribute tounctional development of the carotid body. Detailed ultrastructualhanges in the carotid body of mice are not currently available.

. Genetic factors and the carotid body development

The use of a knockout mouse provides us with a very useful toolo understand the role of a certain gene and its product in carotidody function (Table 2). Regarding the developmental aspects ofhe carotid body, Kameda et al. have shown that Hoxa3 (Kamedat al., 2002) and Mash1 (Ascl1) (Kameda, 2005) are required for theormation of the carotid body. Gdnf and Bdnf are also required forormal development of petrosal chemosensory neurons (Ericksont al., 2001). Other studies listed in Table 2 clearly provided criticalnformation of genetic factors in chemosensory function. For exam-le, homozygous or heterozygous gene knockout of Hif1a, Kcnk3,cnk9, Drd2, Cth, or P2 × 2 decreased carotid chemoreceptor neu-al response to hypoxia. However, many knockout mice die duringre- and perinatal periods (Turgeon and Meloche, 2009), and there-ore, except for agenesis of the carotid body, knockout mice do notlways provide useful information for understanding the postna-al development of carotid body function and morphology. In thisegard, inbred strains of mice may consist of better models to revealhe genetic aspects of carotid body development.

Previous studies identified two strains of inbred mice, DBA/2Jnd A/J, as high and low responders to hypoxia, respectivelyTankersley et al., 1994; Rubin et al., 2003; Campen et al., 2004,005). Further, we have found differences in the carotid body struc-ure (Yamaguchi et al., 2003), as well as hypoxic sensitivity of theB (Otsubo et al., 2011), between these two strains. We can assumehat these differences are a direct result of genetics. Our study hasemonstrated differences in steady-state gene expression in thearotid body between the 4-week-old DBA/2J and A/J mice (Balbir

rophic factors of the carotid body are significantly lower in the A/J

& Neurobiology 185 (2013) 20– 29 25

mice than the DBA/2J mice. Our microarray analysis, together withreal-time RT-PCR, identified many genes that fit these categoriesand were differentially expressed between the carotid bodies ofthe two strains. The results indicate that many genes are involvedin the development of the carotid body and associate with hypoxicchemotransduction. All of these genes could function in concert, orindependently, during the early stages of life to establish functionalcarotid body chemotransduction processes.

In a subsequent study (Kostuk et al., 2012), we focusedon glial cell line-derived neurotrophic factor (Gdnf), distal-lesshomeobox-2 (Dlx2), homeobox msh-like-2 (Msx2), and paired-likehomeobox-2b (Phox2b) which are more expressed in the carotidbody of the 4-week-old DBA/2J mice. Initially, we assumed thatGdnf, its receptor Gfr-�1, and the Ret pathway were responsiblefor the differential growth between these two strains (see Fig. 4).However, in contrast to the carotid bodies of 4 weeks old mice, Gdnfand its receptors were not different between the two strains at 7,10 and 14 days of age when divergence of the morphological straindifferences became apparent. The only gene that showed consis-tent differences with various comparison methods was Msx2. Msx2gene was significantly less expressed in the carotid body of P7 A/Jmice compared with that of DBA/2J mice. Msx2 plays a role in thesurvival, differentiation, and proliferation of cranial neural crestcells (Ishii et al., 2003, 2005), cardiac neural crest cells (Chen et al.,2007), as well as teeth, hair follicles, and bones of the skull (Satokataet al., 2000). Thus it is tempting to suggest that Msx2 may playan important role in the proliferation and survival of glomus cells.Future studies using knockout mice may provide some insights asto whether and how Msx2 contributes to the morphological devel-opment of the carotid body. An alternative approach would be anaddition of other strains with the use of a multifactorial design,which can take a full advantage of using inbred strains (Festing,2010). As shown in Fig. 5, the carotid body of C57BL/6 appears toshow different growth pattern from the carotid bodies of DBA/2Jand A/J strains. Checking a panel of genes related to neural tissuegrowth at the time points when these carotid bodies show differentgrowth may add some new information in the postnatal growth ofthe carotid body.

7. Neonatal programming

Geographical and epidemiological studies indicate thatintrauterine development closely correlates with cardiovas-cular diseases in later life (Barker, 2007). Recent studies havefurther revealed the association of perinatal developmental condi-tions to diabetes, obesity, hypertension and even cancer (Oken andGillman, 2003; Tamashiro and Moran, 2010; Calkins and Devaskar,2011). It appears that this is not limited to metabolic diseases.Inflammation during the neonatal period has been shown tohave long-term effects on immune function (Spencer et al., 2010)and neuroendocrine function (Kentner and Pittman, 2010). Thecarotid body does not escape the long-term effects of early-lifeevents. For example, hyperoxia during perinatal periods impairsthe normal development of the carotid body (Erickson et al., 1998;Prieto-Lloret et al., 2004; Dmitrieff et al., 2012) and results ininhibited respiratory responses to hypoxia and asphyxia in adult-hood (Bisgard et al., 2003; Bavis and Mitchell, 2008). In particular,prolonged hyperoxia irreversibly affects carotid body function:when rats are reared in 60% hyperoxia, the hypoxic response ofcarotid chemoreceptor neural activity in adulthood is severelyattenuated (Bisgard et al., 2003; Prieto-Lloret et al., 2004). The

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Table 2Gene knockout and chemoreceptor function.

Gene Effects of gene deletion Reference

Gene name Alternative name Carotid chemoreceptorneural activity

Breathing Other effects

Basal Hypoxia Basal Hypoxia

Putative chemosensors Cybb gp91-phox NT NT ↔ ↔ [Ca2+]i ↔ Roy et al. (2000)

Nox2 ↔ ↔ NT NT K current inhibition ↔, [Ca2+]i ↔ He et al. (2002)

sLTF ↓ Peng et al. (2009)

Ncf1 P47-phox ↑ ↑ ↑ Sanders et al. (2002)

Hmox2 Hemoxygenase 2 NT NT NT NT CA release ↔ Ortega-Saenz et al. (2006)mRNA (Cyc ↑, TH ↑. Slo1↓)Volume ↑BK channel activity ↔

Hif1a Hif1-alpha ↔ ↓ (+/−) ↔ (+/−) ↔ (+/−) Morphology ↔ (+/−) Kline et al. (2002)↓ Hyperoxia (+/−)

Epas1 Hif2a ↔ ↑ (+/−) Irregular (+/−) ↑ (+/−) Morphology ↔ (+/−) Peng et al. (2011)Apnea ↑ CO2 response ↔ (+/−) (CCNA)

Sdhd Sdh4 NT NT NT NT CA release ↑ (+/−) Piruat et al. (2004)TH+ cells in the CB ↑ (+/−)K current ↔ (+/−)

K channels Kcna1 Kv1.1 ↔ ↑ ↔ ↑ Kline et al. (2005)

Kcnk3 Task1 ↔ ↓ ↔ ↓ CO2 response ↓ (CCNA) Trapp et al. (2008)

NT NT NT NT Volume ↔ Ortega-Saenz et al. (2010)Membrane potential ↔K current, Ca current ↔CA release ↔

Kcnk9 Task3 ↑ ↓ ↔ ↔ CO2 response ↓ (CCNA) Trapp et al. (2008)

NT NT NT NT Volume ↔ Ortega-Saenz et al. (2010)Membrane potential ↓K current, Ca current ↓CA release (basal) ↑

Kcnj16 Kir5.1 ↔ ↔ ↔ ↓ CO2 response ↓ Trapp et al. (2011)

Neurotransmitters Nos3 Enos NT NT Anesthetized ↓ Anesthetized ↓ GC ↑ Kline et al. (2000)

Synthesizing enzymes/receptors NT NT Awake ↔ Awake ↓ CO2 response ↔ (breathing)

Tacr1 Nk1r ↔ ↔ NT NT CA content ↔ Rigual et al. (2002)CA secretion ↔

Drd2 D2r ↔ ↓ ↔ ↔ CA release ↑ Prieto-Lloret et al. (2007)

Cth Cystathionase ↔ ↓ ↓ ↓ CO2 response ↔ Peng et al. (2010)

P2rx2 P2 × 2 ↔ ↓ ↔ ↓ Rong et al. (2003)P2rx3 P2 × 3 ↔ ↔ ↔ ↔

↔ (dbl−/−) ↓↓ (dbl−/−) ↔ ↓ (dbl−/−) CO2 response ↔ (breathing)

Transcription factors Hoxa3 Hox1e NT NT NT NT Defect of CB formation Kameda et al. (2002)

Ascl1 Mash1 NT NT NT NT Defect of glomus cells Kameda (2005)

Mecp2 NT NT ↔ ↑ TH staining (CB, PG) ↓ Roux et al. (2008)

Phox2b Pmx2b NT NT ↔ (+/−) ↔ (+/−) Volume ↓ (fetal), then degenerate Dauger et al. (2003)Posthypoxic apnea ↑ (+/−) No TH staining

CO2 response ↓ (breathing @ P2)PG ↓

Nr4a2 Nurr1 NT NT ↓, Apnea ↑ ↓ Maximum diameter, TH cells ↔ Nsegbe et al. (2004)

Others Bdnf NT NT NT NT PG ↓ Erickson et al. (2001)Gdnf NT NT ↓, Apnea ↑ NT PG ↓

PG ↓↓ (dbl−/−)

Frs2 Frs2a NT NT NT NT Defect of CB formation Kameda et al. (2008)

Due to the availability of a large number of knockout mice, the list may not include all published work. Studies which measured only breathing parameters are not included. ↔, no change; ↑, increase; ↓, decrease; NT, not tested;sLTF, long term facilitation of sensory nerve discharge; CA, catecholamine; Cyc, cyclophilin; TH, tyrosine hydroxylase; CB, carotid body; CCNA, carotid chemoreceptor nerve activity: PG, petrosal ganglion.

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Fig. 5. Microphotographs showing the carotid body and a part of the superior cer-vical ganglion (SCG) from three inbred mice at 14 days old (A: DBA/2J strain; B: A/Jstrain; C: C57BL/6 strain). The carotid body is encircled by a solid line. The largestsections of the carotid body in each mouse are presented. A general cellular struc-ttb

aaowmeegb

d

ure of the carotid body of the mouse is very similar to those of large animals, buthe carotid body is much more closely located to the SCG compared to the carotidody of larger animals. Some of the neurons in the SCG are indicated by arrows.

t 2 weeks of age (Kholwadwala and Donnelly, 1992; Donnellynd Doyle, 1994). Although chemoafferent degeneration was alsobserved in hyperoxic treated rats, chemoafferent degenerationas likely caused by the impairment of the carotid body develop-ent and subsequent decreases in neurotrophic factors (Erickson

t al., 1998; Dmitrieff et al., 2011). Thus, it appears that the adverseffects on the carotid body persist into adulthood, if insults are

Rodents have been used as good experimental tools for humanisorders during neonatal periods due to their large litter size,

& Neurobiology 185 (2013) 20– 29 27

a short duration to mature and availability of inbred strains andknockout mice. Several knockout mice have proved to be useful forinvestigating disorders of respiratory control (Gaultier et al., 2006).However, many knockout mice die perinataly, and therefore, it isdifficult to examine carotid body function. In some neonatal disor-ders, alternative approaches would be to apply the insults describedabove to various inbred strains, which may cause dysfunction of thecarotid body. Differential outcomes among inbred strains would bevaluable means to dissect the genetic background of susceptibilityin the affected population.

8. Summary and future directions

Studies examining chemoreceptor development in mice are notplentiful. We have shown that morphological development is ageand strain specific. We assume that functional development of thecarotid body is also age and strain dependent, because carotid bodychemoreceptor function is strain specific. However, a major chal-lenge is the size of the carotid body, and information of carotidbody neural output in neonatal mice is not currently available. Thehypoxic ventilatory response has been studied in neonatal mice, butit is uncertain if the response truly reflects the carotid body func-tion. A careful comparison of hypoxic responses of chemoreceptorneural output and ventilation in rats suggests that the hypoxic ven-tilatory response cannot be used to assess carotid body function inneonatal rats. Whether or not this is also the case in the mouseneeds further investigation. Can we get there? The developmentof technology is amazingly fast. For example, measuring the fetalleft ventricular pressure is now possible (Le et al., 2012), whichwe could not imagine 10 years ago. Thus, hopefully, we will soonestablish techniques for measuring carotid chemoreceptor neuralactivity from young mice. Then, we can investigate developmentalaspects of not only hypoxia but also other natural and pharmaco-logical stimuli. Further, we would like to emphasize that fastidiouscare and careful considerations are required for obtaining tissues.Because of the small size of the carotid body and the proximity toother tissues, in particular the superior cervical ganglion (see Fig. 5),the contamination or misrepresentation of the tissues by personnelwho are not-well-trained can easily occur. We may need to developsome standards to ensure proper handling of the tissues. These mayinclude special markers which are expressed in the carotid body butnot in the superior cervical ganglion.

Because many knockout mice die during pre- and perinatal peri-ods, various inbred strains should be used to understand geneticmechanisms for the postnatal development of the carotid body.We still have not taken a full advantage of the controlled geneticsof inbred strains. Also, development of targeted gene deletion inthe carotid body is a critical alternate approach.

Acknowledgments

This work was supported by HL81345 and AHA09GRAN2080158. Luis E. Pichard was supported by F31HL096450.

References

Aizenfisz, S., Dauger, S., Durand, E., Vardon, G., Levacher, B., Simonneau, M., Pach-nis, V., Gaultier, C., Gallego, J., 2002. Ventilatory responses to hypercapnia andhypoxia in heterozygous c-ret newborn mice. Respiratory Physiology and Neu-robiology 131, 213–222.

Arata, S., Amano, K., Yamakawa, K., Arata, A., 2010. Central respiratory failure ina mouse model depends on the genetic background of the host. Advances inExperimental Medicine and Biology 669, 21–24.

Balbir, A., 2008. The development of respiratory control and hypoxic sensitivity

Balbir, A., Lande, B., Fitzgerald, R.S., Polotsky, V., Mitzner, W., Shirahata, M., 2008.Behavioral and respiratory characteristics during sleep in neonatal DBA/2 J andA/J mice. Brain Research 1241, 84–91.

2 siology

B

B

B

B

B

B

B

B

B

B

B

B

B

C

C

C

C

C

C

C

C

C

C

CD

D

D

D

D

D

D

8 M. Shirahata et al. / Respiratory Phy

albir, A., Lee, H., Okumura, M., Biswal, S., Fitzgerald, R.S., Shirahata, M., 2007. Asearch for genes that may confer divergent morphology and function in thecarotid body between two strains of mice. American Journal of Physiology. LungCellular and Molecular Physiology 292, L704–L715.

amford, O.S., Sterni, L.M., Wasicko, M.J., Montrose, M.H., Carroll, J.L., 1999. Postnatalmaturation of carotid body and type I cell chemoreception in the rat. AmericanJournal of Physiology 276, L875–L884.

arker, D.J., 2007. The origins of the developmental origins theory. Journal of InternalMedicine 261, 412–417.

avis, R.W., Mitchell, G.S., 2008. Long-term effects of the perinatal environment onrespiratory control. Journal of Applied Physiology 104, 1220–1229.

isgard, G.E., Olson Jr., E.B., Wang, Z.Y., Bavis, R.W., Fuller, D.D., Mitchell, G.S., 2003.Adult carotid chemoafferent responses to hypoxia after 1, 2, and 4 wk of post-natal hyperoxia. Journal of Applied Physiology 95, 946–952.

issonnette, J.M., Knopp, S.J., 2001. Developmental changes in the hypoxic ventila-tory response in C57BL/6 mice. Respiration Physiology 128, 179–186.

lain, G.M., Smith, C.A., Henderson, K.S., Dempsey, J.A., 2009. Contribution of thecarotid body chemoreceptors to eupneic ventilation in the intact, unanes-thetized dog. Journal of Applied Physiology 106, 1564–1573.

lain, G.M., Smith, C.A., Henderson, K.S., Dempsey, J.A., 2010. Peripheral chemore-ceptors determine the respiratory sensitivity of central chemoreceptors to CO2.The Journal of Physiology 588, 2455–2471.

lanco, C.E., Dawes, G.S., Hanson, M.A., McCooke, H.B., 1984. The response to hypoxiaof arterial chemoreceptors in fetal sheep and new-born lambs. Journal of Phys-iology (London) 351, 25–37.

lanco, C.E., Hanson, M.A., McCooke, H.B., 1988. Effects on carotid chemoreceptorresetting of pulmonary ventilation in the fetal lamb in utero. Journal of Devel-opmental Physiology 10, 167–174.

olle, T., Lauweryns, J.M., Lommel, A.V., 2000. Postnatal maturation of neuroep-ithelial bodies and carotid body innervation: a quantitative investigation in therabbit. Journal of Neurocytology 29, 241–248.

oyd, J.D., 1937. The Development of the Human Carotid Body. Contributions toEmbryology. Carnegie Institution of Washington, Baltimore, MD, pp. 1–31.

urton, M.D., Kawashima, A., Brayer, J.A., Kazemi, H., Shannon, D.C., Schuchardt, A.,Costantini, F., Pachnis, V., Kinane, T.B., 1997. RET proto-oncogene is importantfor the development of respiratory CO2 sensitivity. Journal of the AutonomicNervous System 63, 137–143.

alkins, K., Devaskar, S.U., 2011. Fetal origins of adult disease. Current Problems inPediatric and Adolescent Health Care 41, 158–176.

ampen, M.J., Tagaito, Y., Jenkins, T.P., Balbir, A., O’Donnell, C.P., 2005. Heart ratevariability responses to hypoxic and hypercapnic exposures in different mousestrains. Journal of Applied Physiology 99, 807–813.

ampen, M.J., Tagaito, Y., Li, J., Balbir, A., Tankersley, C.G., Smith, P., Schwartz, A.,O’Donnell, C.P., 2004. Phenotypic variation in cardiovascular responses to acutehypoxic and hypercapnic exposure in mice. Physiological Genomics 20, 15–20.

arroll, J.L., 2003. Plasticity in respiratory motor control – invited review: devel-opmental plasticity in respiratory control. Journal of Applied Physiology 94,375–389.

arroll, J.L., Bamford, O.S., Fitzgerald, R.S., 1993. Postnatal maturation of carotidchemoreceptor responses to O2 and CO2 in the cat. Journal of Applied Physiology75, 2383–2391.

arroll, J.L., Fitzgerald, R.S., 1993. Carotid chemoreceptor responses to hypoxia andhypercapnia in developing kittens. Advances in Experimental Medicine and Biol-ogy 337, 387–391.

hen, Y.H., Ishii, M., Sun, J., Sucov, H.M., Maxson Jr., R.E., 2007. Msx1 and Msx2regulate survival of secondary heart field precursors and post-migratory prolif-eration of cardiac neural crest in the outflow tract. Developmental Biology 308,421–437.

hia, R., Achilli, F., Festing, M.F., Fisher, E.M., 2005. The origins and uses of mouseoutbred stocks. Nature Genetics 37, 1181–1186.

lancy, B., Finlay, B.L., Darlington, R.B., Anand, K.J., 2007. Extrapolating brain devel-opment from experimental species to humans. Neurotoxicology 28, 931–937.

larke, J.A., Daly, M.D., 1985. The volume of the carotid body and periadventitialtype I and type II cells in the carotid bifurcation region of the fetal cat and kitten.Anatomy and Embryology-Berlin 173, 117–127.

ollins, F.S., Rossant, J., Wurst, W., 2007. A mouse for all reasons. Cell 128, 9–13.ahan, A., Nieuwenhuijs, D., Teppema, L., 2007. Plasticity of central chemorecep-

tors: effect of bilateral carotid body resection on central CO2 sensitivity. PLoSMedicine 4, e239.

auger, S., Aizenfisz, S., Renolleau, S., Durand, E., Vardon, G., Gaultier, C., Gallego,J., 2001. Arousal response to hypoxia in newborn mice. Respiration Physiology128, 235–240.

auger, S., Durand, E., Cohen, G., Lagercrantz, H., Changeux, J.P., Gaultier, C., Gallego,J., 2004. Control of breathing in newborn mice lacking the beta-2 nAChR subunit.Acta Physiologica Scandinavica 182, 205–212.

auger, S., Pattyn, A., Lofaso, F., Gaultier, C., Goridis, C., Gallego, J., Brunet, J.F., 2003.Phox2b controls the development of peripheral chemoreceptors and afferentvisceral pathways. Development 130, 6635–6642.

insdale, F., Emery, J.L., Gadsdon, D.R., 1977. The carotid body—a quantitative assess-ment in children. Histopathology 1, 179–187.

mitrieff, E.F., Piro, S.E., Broge Jr., T.A., Dunmire, K.B., Bavis, R.W., 2012. Carotid

mitrieff, E.F., Wilson, J.T., Dunmire, K.B., Bavis, R.W., 2011. Chronic hyperoxia altersthe expression of neurotrophic factors in the carotid body of neonatal rats.Respiratory Physiology and Neurobiology 175, 220–227.

& Neurobiology 185 (2013) 20– 29

Donnelly, D.F., 2000. Developmental aspects of oxygen sensing by the carotid body.Journal of Applied Physiology 88, 2296–2301.

Donnelly, D.F., Doyle, T.P., 1994. Developmental changes in hypoxia-induced cate-cholamine release from rat carotid body, in vitro. Journal of Physiology (London)475, 267–275.

Donnelly, D.F., Rigual, R., 2000. Single-unit recordings of arterial chemorecep-tors from mouse petrosal ganglia in vitro. Journal of Applied Physiology 88,1489–1495.

Durand, E., Dauger, S., Pattyn, A., Gaultier, C., Goridis, C., Gallego, J., 2005. Sleep-disordered breathing in newborn mice heterozygous for the transcriptionfactor Phox2b. American Journal of Respiratory and Critical Care Medicine 172,238–243.

Enhorning, G., van Schaik, S., Lundgren, C., Vargas, I., 1998. Whole-body plethys-mography, does it measure tidal volume of small animals? Canadian Journal ofPhysiology and Pharmacology 76, 945–951.

Erickson, J.T., Brosenitsch, T.A., Katz, D.M., 2001. Brain-derived neurotrophic factorand glial cell line-derived neurotrophic factor are required simultaneously forsurvival of dopaminergic primary sensory neurons in vivo. Journal of Neuro-science 21, 581–589.

Erickson, J.T., Mayer, C., Jawa, A., Ling, L., Olson Jr., E.B., Vidruk, E.H., Mitchell, G.S.,Katz, D.M., 1998. Chemoafferent degeneration and carotid body hypoplasia fol-lowing chronic hyperoxia in newborn rats. Journal of Physiology (London) 509(Pt 2), 519–526.

Evans, H.E., Sack, W.O., 1973. Prenatal development of domestic and laboratorymammals: growth curves, external features and selected references. ZentralblVeterinarmed C 2, 11–45.

Festing, M.F., 2010. Inbred strains should replace outbred stocks in toxicology, safetytesting, and drug development. Toxicologic Pathology 38, 681–690.

Gauda, E.B., Carroll, J.L., Donnelly, D.F., 2009. Developmental maturation ofchemosensitivity to hypoxia of peripheral arterial chemoreceptors—invited arti-cle. Advances in Experimental Medicine and Biology 648, 243–255.

Gaultier, C., Gallego, J., 2008. Neural control of breathing: insights from geneticmouse models. Journal of Applied Physiology 104, 1522–1530.

Gaultier, C., Matrot, B., Gallego, J., 2006. Transgenic models to study disorders ofrespiratory control in newborn mice. ILAR Journal 47, 15–21.

He, L., Chen, J., Dinger, B., Sanders, K., Sundar, K., Hoidal, J., Fidone, S., 2002. Char-acteristics of carotid body chemosensitivity in NADPH oxidase-deficient mice.American Journal of Physiology. Cell Physiology 282, C27–C33.

Heath, D., Smith, P., 1992. Diseases of the Human Carotid Body. Springer-Verlag,London.

Hervonen, A., Korkala, O., 1972. Fine structure of the carotid body of themidterm human fetus. Zeitschrift fur Anatomie und Entwicklungsgeschichte138, 135–144.

Holley, H.S., Behan, M., Wenninger, J.M., 2012. Age and sex differences in the ven-tilatory response to hypoxia and hypercapnia in awake neonatal, pre-pubertaland young adult rats. Respiratory Physiology and Neurobiology 180, 79–87.

Ishii, M., Han, J., Yen, H.Y., Sucov, H.M., Chai, Y., Maxson Jr., R.E., 2005. Combineddeficiencies of Msx1 and Msx2 cause impaired patterning and survival of thecranial neural crest. Development 132, 4937–4950.

Ishii, M., Merrill, A.E., Chan, Y.S., Gitelman, I., Rice, D.P., Sucov, H.M., Maxson Jr.,R.E., 2003. Msx2 and Twist cooperatively control the development of the neuralcrest-derived skeletogenic mesenchyme of the murine skull vault. Development130, 6131–6142.

Kameda, Y., 2005. Mash1 is required for glomus cell formation in the mouse carotidbody. Developmental Biology 283, 128–139.

Kameda, Y., Ito, M., Nishimaki, T., Gotoh, N., 2008. FRS2 alpha 2F/2F mice lack carotidbody and exhibit abnormalities of the superior cervical sympathetic ganglionand carotid sinus nerve. Developmental Biology 314, 236–247.

Kameda, Y., Nishimaki, T., Takeichi, M., Chisaka, O., 2002. Homeobox gene hoxa3 isessential for the formation of the carotid body in the mouse embryos. Develop-mental Biology 247, 197–209.

Kariya, I., Nakjima, T., Ozawa, H., 1990. Ultrastructural study on cell differentiationof the rabbit carotid body. Archives of Histology and Cytology 53, 245–258.

Karlsson, K.A., Blumberg, M.S., 2002. The union of the state: myoclonic twitching iscoupled with nuchal muscle atonia in infant rats. Behavioral Neuroscience 116,912–917.

Karlsson, K.A., Kreider, J.C., Blumberg, M.S., 2004. Hypothalamic contribution tosleep–wake cycle development. Neuroscience 123, 575–582.

Kazemian, P., Stephenson, R., Yeger, H., Cutz, E., 2001. Respiratory control in neonatalmice with NADPH oxidase deficiency. Respiration Physiology 126, 89–101.

Kentner, A.C., Pittman, Q.J., 2010. Minireview: early-life programming by inflamma-tion of the neuroendocrine system. Endocrinology 151, 4602–4606.

Kholwadwala, D., Donnelly, D.F., 1992. Maturation of carotid chemoreceptor sen-sitivity to hypoxia: in vitro studies in the newborn rat. Journal of Physiology(London) 453, 461–473.

Kline, D.D., Buniel, M.C., Glazebrook, P., Peng, Y.J., Ramirez-Navarro, A., Prabhakar,N.R., Kunze, D.L., 2005. Kv1.1 deletion augments the afferent hypoxic chemosen-sory pathway and respiration. Journal of Neuroscience 25, 3389–3399.

Kline, D.D., Peng, Y.J., Manalo, D.J., Semenza, G.L., Prabhakar, N.R., 2002. Defectivecarotid body function and impaired ventilatory responses to chronic hypoxiain mice partially deficient for hypoxia-inducible factor 1alpha. Proceedings

Kline, D.D., Yang, T., Premkumar, D.R., Thomas, A.J., Prabhakar, N.R., 2000. Bluntedrespiratory responses to hypoxia in mutant mice deficient in nitric oxidesynthase-3. Journal of Applied Physiology 88, 1496–1508.

siology

K

K

K

K

L

L

M

M

M

N

N

OO

O

O

P

P

P

P

P

P

P

P

R

R

M. Shirahata et al. / Respiratory Phy

ondo, H., 1975. A light and electron microscopic study on the embryonic develop-ment of the rat carotid body. American Journal of Anatomy 144, 275–293.

ondo, H., 1976. An electron microscopic study on the development of synapses inthe rat carotid body. Neuroscience Letters 3, 197–200.

ostuk, E.W., Balbir, A., Fujii, K., Fujioka, A., Pichard, L.E., Shirahata, M., 2012. Diver-gent postnatal development of the carotid body in DBA/2J and A/J strains of mice.Journal of Applied Physiology 112, 490–500.

rimsky, W.R., Leiter, J.C., 2005. Physiology of breathing and respiratory controlduring sleep. Seminars in Respiratory and Critical Care Medicine 26, 5–12.

e, V.P., Kovacs, A., Wagenseil, J.E., 2012. Measuring left ventricular pressure inlate embryonic and neonatal mice. The Journal of Visualized Experiments 60,http://dx.doi.org/10.3791/3756, pii: 3756.

iu, Q., Lowry, T.F., Wong-Riley, M.T., 2006. Postnatal changes in ventilation duringnormoxia and acute hypoxia in the rat: implication for a sensitive period. TheJournal of Physiology 577, 957–970.

ortola, J.P., Noworaj, A., 1985. Breathing pattern and growth: comparative aspects.Journal of Comparative Physiology. B, Biochemical, Systemic, and EnvironmentalPhysiology 155, 171–176.

ortola, J.P., Rezzonico, R., Lanthier, C., 1989. Ventilation and oxygen consumptionduring acute hypoxia in newborn mammals: a comparative analysis. RespirationPhysiology 78, 31–43.

ulligan, E., 1991. Discharge properties of carotid bodies: developmental aspects.In: Haddad, G.G., Farber, J.P. (Eds.), Developmental Neurobiology of Breathing.Marcel Dekker, Inc., New York, pp. 321–340.

segbe, E., Wallen-Mackenzie, A., Dauger, S., Roux, J.C., Shvarev, Y., Lagercrantz,H., Perlmann, T., Herlenius, E., 2004. Congenital hypoventilation and impairedhypoxic response in Nurr1 mutant mice. The Journal of Physiology 556, 43–59.

urse, C.A., Buttigieg, J., Thompson, R., Zhang, M., Cutz, E., 2006. Oxygen sensing inneuroepithelial and adrenal chromaffin cells. Novartis Foundation Symposium272, 106–114.

ken, E., Gillman, M.W., 2003. Fetal origins of obesity. Obesity Research 11, 496–506.rtega-Saenz, P., Levitsky, K.L., Marcos-Almaraz, M.T., Bonilla-Henao, V., Pascual, A.,

Lopez-Barneo, J., 2010. Carotid body chemosensory responses in mice deficientof TASK channels. Journal of General Physiology 135, 379–392.

rtega-Saenz, P., Pascual, A., Gomez-Diaz, R., Lopez-Barneo, J., 2006. Acute oxy-gen sensing in heme oxygenase-2 null mice. Journal of General Physiology 128,405–411.

tsubo, T., Kostuk, E.W., Balbir, A., Fujii, K., Shirahata, M., 2011. Differential expres-sion of large-conductance Ca-activated K channels in the carotid body betweenDBA/2J and A/J strains of mice. Frontiers in Cellular Neuroscience 5, 19.

ack, A.I., Galante, R.J., Maislin, G., Cater, J., Metaxas, D., Lu, S., Zhang, L., Von Smith, R.,Kay, T., Lian, J., Svenson, K., Peters, L.L., 2007. Novel method for high-throughputphenotyping of sleep in mice. Physiological Genomics 28, 232–238.

eng, Y.J., Nanduri, J., Khan, S.A., Yuan, G., Wang, N., Kinsman, B., Vaddi, D.R., Kumar,G.K., Garcia, J.A., Semenza, G.L., Prabhakar, N.R., 2011. Hypoxia-inducible fac-tor 2alpha (HIF-2alpha) heterozygous-null mice exhibit exaggerated carotidbody sensitivity to hypoxia, breathing instability, and hypertension. Proceed-ings of the National Academy of Sciences of the United States of America 108,3065–3070.

eng, Y.J., Nanduri, J., Raghuraman, G., Souvannakitti, D., Gadalla, M.M., Kumar, G.K.,Snyder, S.H., Prabhakar, N.R., 2010. H2S mediates O2 sensing in the carotid body.Proceedings of the National Academy of Sciences of the United States of America107, 10719–10724.

eng, Y.J., Nanduri, J., Yuan, G., Wang, N., Deneris, E., Pendyala, S., Natarajan, V.,Kumar, G.K., Prabhakar, N.R., 2009. NADPH oxidase is required for the sen-sory plasticity of the carotid body by chronic intermittent hypoxia. Journal ofNeuroscience 29, 4903–4910.

epper, D.R., Landauer, R.C., Kumar, P., 1995. Postnatal development of CO2–O2

interaction in the rat carotid body in vitro. Journal of Physiology (London) 485,531–541.

iruat, J.I., Pintado, C.O., Ortega-Saenz, P., Roche, M., Lopez-Barneo, J., 2004. Themitochondrial SDHD gene is required for early embryogenesis, and its partialdeficiency results in persistent carotid body glomus cell activation with fullresponsiveness to hypoxia. Molecular and Cellular Biology 24, 10933–10940.

rieto-Lloret, J., Caceres, A.I., Obeso, A., Rocher, A., Rigua, lR., Agapito, M.T., Bus-tamante, R., Castaneda, J., Perez-Garcia, M.T., Lopez-Lopez, J.R., Gonzalez, C.,2004. Ventilatory responses and carotid body function in adult rats perinatallyexposed to hyperoxia. Journal of Physiology (London) 554, 126–144.

rieto-Lloret, J., Donnelly, D.F., Rico, A.J., Moratalla, R., Gonzalez, C., Rigual, R.J.,2007. Hypoxia transduction by carotid body chemoreceptors in mice lackingdopamine D(2) receptors. Journal of Applied Physiology 103, 1269–1275.

enolleau, S., Dauger, S., Autret, F., Vardon, G., Gaultier, C., Gallego, J., 2001a. Matura-tion of baseline breathing and of hypercapnic and hypoxic ventilatory responses

enolleau, S., Dauger, S., Vardon, G., Levacher, B., Simonneau, M., Yanagisawa, M.,Gaultier, C., Gallego, J., 2001b. Impaired ventilatory responses to hypoxia in micedeficient in endothelin-converting-enzyme-1. Pediatric Research 49, 705–712.

& Neurobiology 185 (2013) 20– 29 29

Rigual, R., Almaraz, L., Gonzalez, C., Donnelly, D.F., 2000. Developmental changesin chemoreceptor nerve activity and catecholamine secretion in rabbit carotidbody: possible role of Na+ and Ca2+ currents. Pflugers Archiv 439, 463–470.

Rigual, R., Rico, A.J., Prieto-Lloret, J., de Felipe, C., Gonzalez, C., Donnelly, D.F., 2002.Chemoreceptor activity is normal in mice lacking the NK1 receptor. EuropeanJournal of Neuroscience 16, 2078–2084.

Robinson, D.M., Kwok, H., Adams, B.M., Peebles, K.C., Funk, G.D., 2000. Developmentof the ventilatory response to hypoxia in Swiss CD-1 mice. Journal of AppliedPhysiology 88, 1907–1914.

Rogers, D.C., 1965. The development of the rat carotid body. Journal of Anatomy 99,89–101.

Rong, W., Gourine, A.V., Cockayne, D.A., Xiang, Z., Ford, A.P., Spyer, K.M., Burnstock,G., 2003. Pivotal role of nucleotide P2X2 receptor subunit of the ATP-gated ionchannel mediating ventilatory responses to hypoxia. Journal of Neuroscience23, 11315–11321.

Roux, J.C., Dura, E., Villard, L., 2008. Tyrosine hydroxylase deficit in the chemoaf-ferent and the sympathoadrenergic pathways of the Mecp2 deficient mouse.Neuroscience Letters 447, 82–86.

Roy, A., Rozanov, C., Mokashi, A., Daudu, P., Al mehdi, A.B., Shams, H., Lahiri, S.,2000. Mice lacking in gp91 phox subunit of NAD(P)H oxidase showed glomuscell [Ca2+]i and respiratory responses to hypoxia. Brain Research 872, 188–193.

Rubin, A.E., Polotsky, V.Y., Balbir, A., Krishnan, J.A., Schwartz, A.R., Smith, P.L., Fitzger-ald, R.S., Tankersley, C.G., Shirahata, M., O’Donnell, C.P., 2003. Differences insleep-induced hypoxia between A/J and DBA/2J mouse strains. American Journalof Respiratory and Critical Care Medicine 168, 1520–1527.

Sanders, K.A., Sundar, K.M., He, L., Dinger, B., Fidone, S., Hoidal, J.R., 2002. Role ofcomponents of the phagocytic NADPH oxidase in oxygen sensing. Journal ofApplied Physiology 93, 1357–1364.

Satokata, I., Ma, L., Ohshima, H., Bei, M., Woo, I., Nishizawa, K., Maeda, T., Takano, Y.,Uchiyama, M., Heaney, S., Peters, H., Tang, Z., Maxson, R., Maas, R., 2000. Msx2deficiency in mice causes pleiotropic defects in bone growth and ectodermalorgan formation. Nature Genetics 24, 391–395.

Schaub, C.D., Tankersley, C., Schwartz, A.R., Smith, P.L., Robotham, J.L., O’Donnell,C.P., 1998. Effect of sleep/wake state on arterial blood pressure in geneticallyidentical mice. Journal of Applied Physiology 85, 366–371.

Schneider, B.F., Norton, S., 1979. Equivalent ages in rat, mouse and chick embryos.Teratology 19, 273–278.

Shea, S.A., 1996. Behavioural and arousal-related influences on breathing in humans.Experimental Physiology 81, 1–26.

Smith, C., 1924. The origin and development of the carotid body. American Journalof Anatomy 34, 87–131.

Smith, P., Scraggs, M., Heath, D., 1993. The development of the nerve network inthe fetal human carotid body and its subsequent function in cardiac disease.Cardioscience 4, 143–149.

Spencer, S.J., Field, E., Pittman, Q.J., 2010. Neonatal programming by neuroimmunechallenge: effects on responses and tolerance to septic doses of lipopolysaccha-ride in adult male and female rats. Journal of Neuroendocrinology 22, 272–281.

Sterba, O., 1995. Staging and ageing of mammalian embryos and fetuses. Acta Vet-erinaria Brno 64, 83–89.

Tagaito, Y., Polotsky, V.Y., Campen, M.J., Wilson, J.A., Balbir, A., Smith, P.L., Schwartz,A.R., O’Donnell, C.P., 2001. A model of sleep-disordered breathing in the C57BL/6Jmouse. Journal of Applied Physiology 91, 2758–2766.

Tamashiro, K.L., Moran, T.H., 2010. Perinatal environment and its influences onmetabolic programming of offspring. Physiology and Behavior 100, 560–566.

Tankersley, C.G., Fitzgerald, R.S., Kleeberger, S.R., 1994. Differential control of ven-tilation among inbred strains of mice. American Journal of Physiology 267,R1371–R1377.

Timmers, H.J., Karemaker, J.M., Wieling, W., Marres, H.A., Folgering, H.T., Lenders,J.W., 2003. Baroreflex and chemoreflex function after bilateral carotid bodytumor resection. Journal of Hypertension 21, 591–599.

Trapp, S., Aller, M.I., Wisden, W., Gourine, A.V., 2008. A role for TASK-1 (KCNK3)channels in the chemosensory control of breathing. Journal of Neuroscience 28,8844–8850.

Trapp, S., Tucker, S.J., Gourine, A.V., 2011. Respiratory responses to hypercapnia andhypoxia in mice with genetic ablation of Kir5.1 (Kcnj16). Experimental Physiol-ogy 96, 451–459.

Turgeon, B., Meloche, S., 2009. Interpreting neonatal lethal phenotypes in mousemutants: insights into gene function and human diseases. Physiological Reviews89, 1–26.

Wang, Z.Y., Bisgard, G.E., 2005. Postnatal growth of the carotid body. RespiratoryPhysiology and Neurobiology 149, 181–190.

Wasicko, M.J., Sterni, L.M., Bamford, O.S., Montrose, M.H., Carroll, J.L., 1999. Resettingand postnatal maturation of oxygen chemosensitivity in rat carotid chemore-

Yamaguchi, S., Balbir, A., Schofield, B., Coram, J., Tankersley, C.G., Fitzgerald, R.S.,O’Donnell, C.P., Shirahata, M., 2003. Structural and functional differences ofthe carotid body between DBA/2J and A/J strains of mice. Journal of AppliedPhysiology 94, 1536–1542.