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Please cite this article in press as: Bairam A, et al., Developmental profile of cholinergic and purinergic traits and receptors in periph-eral chemoreflex pathway in cats, Neuroscience (2007), doi: 10.1016/j.neuroscience.2007.03.034

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EVELOPMENTAL PROFILE OF CHOLINERGIC AND PURINERGICRAITS AND RECEPTORS IN PERIPHERAL CHEMOREFLEX

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. BAIRAM,* V. JOSEPH, Y. LAJEUNESSEND R. KINKEAD

nité de recherche en périnatologie, Centre Hospitalier Universitairee Québec, Hôpital Saint-François d’Assise, Département de Pédiat-ie, Université Laval, Québec, Canada

bstract—This study describes the developmental profile ofpecific aspects of cholinergic and purinergic neurotrans-ission in key organs of the peripheral chemoreflex: the

arotid body (CB), petrosal ganglion (PG) and superior cer-ical ganglion (SCG). Using real time RT-PCR and Westernlot analyses, we assessed both mRNA and protein expres-ion levels for choline-acetyl-transferase (ChAT), nicotiniceceptor (subunits �3, �4, �7, and �2), ATP and purinergiceceptors (P2X2 and P2X3). These analyses were performedn tissue from 1- and 15-day-old, 2-month-old, and adult cats.uring development, ChAT protein expression level in-reased slightly in CB; however, this increase was moremportant in PG and SCG. In CB, mRNA level for �4 nicotiniceceptor subunit decreased during development (90% highern 1-day-old cats than in adults). In the PG, mRNA level for �2

icotinic receptor subunit increased during development80% higher in adults than in 1-day-old cats). In SCG, mRNAor �7 nicotinic receptor levels increased (400% higher indults vs. 1-day-old cats). Conversely, P2X2 receptor protein

evel was not altered during development in CB and de-reased slightly in PG; a similar pattern was observed for the2X3 receptor. Our findings suggest that in cats, age-relatedhanges in cholinergic and purinergic systems (such ashysiological expression of receptor function) are significantithin the afferent chemoreceptor pathway and likely contrib-te to the temporal changes of O2-chemosensitivity duringevelopment. © 2007 IBRO. Published by Elsevier Ltd. Allights reserved.

ey words: carotid body, acetylcholine, ATP, nicotinic recep-ors, purinergic receptors, development.

he process of oxygen sensitivity of carotid body (CB) typechemoreceptor cells is immature at birth. It takes days toeeks to reach full maturity depending on species studied

Carroll, 2003; Bairam and Carroll, 2005). Although theole of cholinergic neurotransmission in CB function is stillebated, most of electrophysiological data obtained toate are consistent with the hypothesis that acetylcholineACh) and ATP are mandatory for neural transmission

Correspondence to: A. Bairam, Centre de Recherche, D0-717, 10,ue de l’Espinay, Quebec (PQ), Canada G1L 3L5. Tel: �1-418-525-402; fax: �1-418-525-4195.-mail address: Aida.bairam@crsfa.ulaval.ca (A. Bairam).bbreviations: ACh, acetylcholine; CB, carotid body; ChAT, choline

scetyl-transferase; PBS-T, PBS with 0.1% Tween-20; P2X, purinergiceceptor; SCG, superior cervical ganglion.

306-4522/07$30.00�0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reseroi:10.1016/j.neuroscience.2007.03.034

1

etween type I cells and carotid sinus nerve endingsNurse and Zhang, 2001; Prabhakar, 2006; Iturriaga et al.,n press; Shirahata et al., 2007). Although both transmittersre co-released in response to hypoxia and increase ca-otid sinus nerve firing rate (Nurse and Zhang, 2001; Prab-akar, 2006; Reyes et al., 2007b), their developmentalattern in the CB is unknown.

ACh acts both on nicotinic and muscarinic receptorsocated on chemosensitive cells and carotid sinus nerveerminals in the CB (Shirahata et al., 1998, 2007; Fitzger-ld, 2000; Bairam et al., 2006). Among the 12 differenticotinic receptors subunits, cultured type I cells from catB are immunopositive for �3, �4 and �2 (Higashi et al.,003), whereas the �7 subunit is present on nerve termi-als surrounding the chemoreceptor cells (Shirahata et al.,998). Moreover, �3, �4, �7 and �2 receptor subunits haveeen identified on some cell bodies of cat petrosal gan-lion also (Shirahata et al., 1998; Fitzgerald, 2000; Shira-ata, 2002). These nicotinic receptors are sensitive to AChgonists or antagonists regardless of their locations (che-oreceptor cells or petrosal ganglion neurons) (Dinger etl., 1985; Nurse and Zhang, 1999; Fitzgerald, 2000; Zhangt al., 2000; Higashi et al., 2003; Varas et al., 2003, 2006;onde and Monteiro, 2006).

Although detailed developmental studies of CB nico-inic receptors and their functional role are still lacking,ome data support an age-dependent functional matura-ion. In an in vitro CB study, carbachol (a full cholinergicgonist) was shown to enhance dopamine release underasal condition in adults but not in 10-day-old rabbits.owever, carbachol enhanced hypoxia-induced dopamine

elease in a larger magnitude in 10-day-old rabbits than indults (Bairam et al., 2001). This observation is partiallyxplained by a lower efficiency of the inhibitory feedbackechanism related to CB M2 receptors in newborn

Bairam et al., 2001), but their expression level did nothange during development (Bairam et al., 2006). Alter-ately, lower CB nicotinic receptors and lower ACh syn-hesis within glomus cells in the newborn than in adultsay be involved.

During hypoxia, ATP released was measured fromither cultured type I cells or from the whole CB in ratButtigieg and Nurse, 2004) where it was shown to en-ance ACh-induced increase of chemosensory activityNurse and Zhang, 2001). In rats, the co-release of ATPnd ACh seems mandatory for a full hypoxic responseZhang et al., 2000) whereas in cats, it does not completelyccount for the hypoxia-induced increase in chemosen-

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eurotransmitter through activation of cell-surface puriner-ic receptors (P2X) (ligand-gated cationic channels, iono-ropic) (Khakh, 2001) and as presynaptic modulatorhrough activation of G protein-coupled, metabotropic P2Yeceptors (Cunha and Ribeiro, 2000). Of the seven knownubunits of P2X receptors (North, 2002), P2X2 appears toontribute to CB chemotransmission since both the che-osensory activity and the ventilatory response to hypoxiare dramatically attenuated in mice lacking P2X2 receptorsompared with wild type or to P2X3 receptors deprivedice (Rong et al., 2003). Both P2X2 and P2X3 receptorsre located in petrosal ganglion neurons and on afferenterve terminals of the sinus nerve in both rats and miceZhang et al., 2000; Prasad et al., 2001; Rong et al., 2003;e et al., 2006).

Overall, these observations raise significant questionsbout the potential age-dependent expression of nicotinicnd purinergic system in the CB. To address this questione sought to determine the developmental pattern of ex-ression of the nicotinic �3, �4, �7 and �2 receptors sub-nits and the purinergic P2X2 and P2X3 receptors. Wesed cats as experimental model because many of theseeceptors have been identified in this species. Otherrgans involved in the chemoreflex pathway, including

he petrosal ganglion (which contains neuronal bodies ofhe carotid sinus nerve), and the superior cervical gan-lion (SCG) (which provides sympathetic innervation to

he CB) were studied also. Since changes in the mRNAxpression level may not be followed by parallelhanges in the protein level (Bairam et al., 2006), weombined real time RT-PCR amplification and Westernlot analyses, when this was technically feasible. ATP

evels were determined using luciferin-luciferase biolu-inescence assays. Our data clearly show that the ex-ression pattern of cholinergic and purinergic systemsvolves during development, these findings may help toetter understand postnatal development of peripheralhemoreceptor functions.

able 1. Oligonucleotide primers selected for real-time PCR and expe

Forward primers Reverse

-3 5=-ATCATCCCCTGCCTGGTCA-3=residues (493–511)

5=-GAGGresidu

-4 5=-GTCCACTTCGGGCTGTCCAT-3=residues (77–96)

5=-GCTTresidu

-7 5=-CAGCCGCTCACCGTCTACTT-3=residues (86–105)

5=-CCATresidu

-2 5=-AGCACTTCCCCTTCGACCA-3=residues (58–76)

5=-TCCAresidu

hAT 5=-ACAGTCACTCCATCCCCACC-3=residues (268–287)

5=-GAGCresidu

2X2 5=-GGTTAGCAACGCCTCCTGTG-3=residues (302–321)

5=-CGAGresidu

2X3 5=-ACCTGGTAAGCCTTCTCGTGTAA-3=residues (394–416)

5=-GGTCresidu

8S 5=-CTGGTACAGTGAAACTGCGAATG-3=residues (53–75)

5=-AGCGresidu

Nicotinic receptors (�-3, �-4, �-7 and �-2); ChAT enzyme; purinerg

Please cite this article in press as: Bairam A, et al., Developmental proeral chemoreflex pathway in cats, Neuroscience (2007), doi: 10.1016/j.n

EXPERIMENTAL PROCEDURES

xperiments were performed on cats from four different ageroups: �1 day, 15 days, 2 months, and 6–8 months old obtainedrom our local animal house facilities. All the experimental proce-ures were approved by the animal ethical committee of our

nstitution, and conformed to the guidelines of the Canadian Com-ittee for Animal Protection. All efforts were made to minimize theumber of animals used and their suffering.

Animal preparation, anesthesia, surgery, and organ collectionere performed as described previously (Bairam et al., 1996,006). Carotid bodies, SCG, and petrosal ganglion were removeduickly and immediately frozen on dry ice before being stored at80 °C.

Four independent pools of carotid bodies, superior cervicalanglia, and petrosal ganglia were collected for each age group.wo pools were used for RNA amplification while the other twoere used for Western blotting and ATP assays. Each pool used0 1-day-old cats (total 40 kittens), 8 15-day-old (total 32 kittens),2-month-old (total 24 cats) and 5 6- to 8-month-old (total 20 adultales).

eal time RT-PCR, semi-quantitative real-timeeverse transcriptase polymerase chain reaction

elative quantification of mRNA expression levels for purinergicnd nicotinic receptors and for ChAT enzyme was assessed usingeal-time PCR amplification. The procedures for total RNA and RTreparation have been previously described (Bairam et al., 1996,006; Joseph and Bairam, 2004). Briefly, aliquots of 2 �l from theesulting single-stranded cDNA products were used along with theppropriate primers for each transcript studied (Table 1). Eachmplification was run in separate wells with 2� SYBR Greenniversal PCR Master Mix containing 100 nM of each specific

orward and reverse gene primers (total 25 �l). PCR amplificationas performed on an ABI Prism 7000 Sequence Detector System

Applied Biosystems, Foster City, CA, USA) according to manu-acturer’s instruction. Amplification conditions were the same forll targets assessed: one cycle at 95 °C for 10 min, 40 cycles at5 °C for 15 s and at 60 °C for 60 s. For each organ, serial dilutionsf 1 �g cDNA (1/10) were used to generate a standard curve forach gene. Amplification of 18S ribosomal RNA was used toormalize data for the difference in starting material, RNA extrac-ion efficiencies, and RT reactions for each sample. Control sam-

gth of the PCR products

Expectedlength (bp)

GeneBank accessionnumber (cat)

GGGATTGTCTCG-3=641)

149 AF411811

GTGCCATTCCTG-3=179)

103 AF411813

AACGAACAGTCT-3=255)

170 DQ777627

GAACTTCATG-3=08)

51 AF411812

GTATTGCTTCATA-3=347)

80 DQ777628

GCTGACCGAC-3=477)

176 DQ777625

CGAGCAGTCC-3=474)

81 DQ777626

CCAAAGGAAC-3=13)

61 AY150542

rs (P2X and P2X ). Primers were chosen using the Primer Express

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les amplified without RT did not yield any amplified signal. A totalf four RT were prepared from the two different RNA pools. Then,hree PCR were performed from each RT giving a total of 12 PCReactions for each amplified gene.

estern blot analysis

embrane (for purinergic receptors) and cytosolic (for ChAT andTP) protein fractions were prepared as described previously

Wang et al., 2002; Bairam et al., 2006). Conditions for tissuesreparation, solutions, homogenization and extraction, proteinoncentration assays, and immunoblotting procedure have beeneported previously (Bairam et al., 2006). For purinergic receptors,0 �g of membrane protein (for CB and petrosal ganglion) or0 �g (for SCG) was used. The protein was first denatured byoiling for 5 min in SDS buffer (75 mM Tris–HCl, pH 6.8, 2% SDS,0% glycerol, 710 mM beta-mercaptoethanol, 0.1% Bromophenollue), electrophoresed by SDS-PAGE on 10% gels, and then

ransferred by electrophoresis overnight onto nitrocellulose mem-ranes (GE Osmonics, Minnetonka, MN, USA). Membranes werehen rinsed in PBS with 0.1% Tween-20 (PBS-T), blocked underhaking for 1 h with 5% fat-free milk in PBS-T at room temperatureo block nonspecific antibody binding. They were then incubated inlocking solution for 2 h at room temperature with specific primaryntibodies (Table 2). After three washes in PBS-T under shaking,he membranes were incubated again for 1 h at room temperaturen horseradish peroxidase–conjugated secondary antibody (Table) in blocking solution. After three times washing with PBS-T,pecific bands were visualized by enhanced Chemiluminescenceeagent Plus (GE Healthcare, Montreal, QC, Canada). A similarrocedure was followed using 10 �g of cytoplasmic protein frac-

ion to identify the ChAT protein with specific primary and second-ry antibodies (Table 2). Specific bands were visualized by en-anced Chemiluminescence Reagent (GE Healthcare).

Membranes were exposed to X-ray film (Fuji, Le Groupehristie, Montreal, QC, Canada) and the intensity of the signalas quantified by densitometry using the Image Analyzer Soft-are Program Quantity One 4.2 (Bio-Rad, Mississauga, ON, Can-da). Because these antibodies had not been previously tested inats, preliminary experiments using samples from adult rat and catCG were used to test the efficiency of each antibody in bothpecies. Signals at the expected molecular weight were reportedoth in rats and cats (data not shown). These signals were 75, 60,8, and 43 kDa for P2X2, P2X3, choline acetyl-transferase (ChAT),nd actin, respectively. Then, specific control experiments forach tested protein were done regularly (see results). For ChATntibody, the specificity of signals was tested in control experi-ents using either recombinant ChAT protein (Chemicon, Te-ecula, CA, USA) or by omitting to add the specific primaryntibody, as shown in Fig. 2. For purinergic receptors, controlxperiments were done using either fusion protein antigen inhich each of the specific primary antibodies was pre-incubatedith its specific antigen (Alomone, Jerusalem, Israel) in PBS–1%SA for 1 h, or by omitting the addition of the specific primaryntibody as shown in Figs. 7 and 8.

able 2. Antibodies used for Western blot analyses and their sources

ntigen Primary antibo

hAT-Human placental enzyme Goat polyclonaTemecula, C

2X2 receptor-17 amino acid synthetic peptidesderived from C-terminus of rat

Rabbit polyclonJerusalem, I

2X3 recptor-16 amino acid synthetic peptidesderived from C-terminus of rat

Rabbit polyclonJerusalem, I

ctin-Cytoskeletal proteins from chicken Mouse monocl

Please cite this article in press as: Bairam A, et al., Developmental proeral chemoreflex pathway in cats, Neuroscience (2007), doi: 10.1016/j.n

TP assays

ytosolic ATP was measured with a luciferin-luciferase biolumi-escence based-method, as used previously (Acker and Star-

inger, 1984; Obeso et al., 1985; Buttigieg and Nurse, 2004). ATPas assessed using ATP determination kit following manufacturerescription (Molecular Probes Invitrogen, OR, USA); all manipu-

ations were performed on melting ice. Standard curves were firstrepared at known concentrations of ATP with luciferin-luciferaseeagent by serial dilution from 1 nM to 1000 nM ATP. Each dosef ATP was measured in triplicate to calculate the final mean pointer point to establish the standard curve. Bioluminescence wasxpressed in relative light units (RLU) and measured in a biolu-inescence analyzer (Mini Lumat LB 9506, Mandel Scientific Co.,ontreal, QC, Canada) for 40 s. All standard curves were consis-

ent among them (see example Fig. 4). In our samples, ATP waseasured by adding 100 �l of luciferin-luciferase mix solution to0 �g cytosolic protein. Six ATP samples were performed fromach pool of organ studied. Hence the final ATP level for each agend each organ represents the mean of 12 ATP measurements.

ata analyses and statistics

or RT-PCR, the relative expression level of each transcript wasrst normalized to the expression level of 18S obtained from theame cDNA sample following the standard curve method instruc-ions given by the manufacturer (User Bulletin, version 2, ABIRISM 7700 sequence detection system). Then, the mean nor-alized gene expression level and standard deviation for each

ranscript studied at each age were calculated. The level obtainedor 1-day-old cats was arbitrarily given the unit of 1 and used as aoint of reference for comparisons with other age groups. Forach transcript, comparisons were made between ages but notetween organs. The mean relative expression level for eachranscript at each age represents the mean of 12 PCR reactions.

The variability of 18S expression level in our experiment wasess than 5%; nonetheless, any variability in the expression levelsf each receptor mRNA studied within 10% of control was notiven a significant interpretation even if the P value was below theignificant threshold (�0.05).

For Western blot, the membrane receptor purinergic proteins well as the cytoplasmic ChAT protein was first normalized tootal actin which is not influenced by age (Cheung et al., 1987).hen the level obtained in 1-day-old cats was arbitrarily given thenit of 1 for subsequent comparisons between ages. The variabil-

ty of membrane actin or cytosolic actin in our samples was lesshan 6% but was considered for statistical analyses as mentionedor 18S. The relative mean protein level for purinergic receptorsnd ChAT enzyme at each age studied represented the mean ofight Western blot analyses (four Western from one pool of or-ans). A similar calculation was used when ChAT expression levelr purinergic protein level was determined between organs. Inhese experiments, the ChAT or receptor level in the CB wasrbitrarily given the unit of 1.

Secondary antibody

Chemicon, Mouse anti-goat, 1/5000, Santa Cruz,Santa Cruz, CA, USA

0, Alomone, Goat anti-rabbit, 1/50000, JacksonImmunoresearch, West Grove, PA, USA

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For the ATP assay, the mean ATP content represents theean value of 12 cytoplasmic measures (six from each pool ofrgans) for each age in each organ. The mean ATP content thatas obtained at 1 day old was given an arbitrary unit of 1 and used

o determine the developmental pattern in each organ.All statistical analysis was done using ANOVA (age as

rouping variable for each organ) followed when significant bypost-ANOVA Fisher’s PLSD to determine the specific signif-

cant level of difference between ages (StatView 4.5). All datare expressed as mean�S.D. Differences were consideredignificant at P�0.05.

RESULTS

holinergic system

Developmental pattern of ChAT mRNAs and proteinxpression. Both mRNA and cytosolic protein fraction of

he ChAT enzyme were identified at all ages (Fig. 1; rep-esentative Western blots are shown at the top of histo-rams B, D and F).

In the CB, the relative mRNA level estimated by realime RT-PCR analyses (Fig. 1A) decreased by 0.4 units in5-day-old cats and was 2.4 and 2 times higher in-month-old and adult cats, respectively in comparison

ig. 1. Developmental pattern of mRNA expression and protein levels

Please cite this article in press as: Bairam A, et al., Developmental proeral chemoreflex pathway in cats, Neuroscience (2007), doi: 10.1016/j.n

ith 1-day-old cats. However, the protein level increasedith age (Fig. 1B) being 1.6 times higher in 2-month-oldnd adult cats relatively to 1- and 15-day-old cats.

In the petrosal ganglion, the relative level of ChATRNA transcript in 15 day, 2 month and adult cats de-

reased to 0.7–0.6 times compared with 1-day-old catsFig. 1C). Interestingly, the protein level did not follow thehanges observed in the mRNA levels (Fig. 1D). Therotein level in 15-day-old cats was 2.3 times higher than-day-old and 3.9 times higher in 2-month-old and adultats compared with 1-day-old.

In the SCG, as for petrosal ganglion, mRNA level in 15ay, 2 month and adult cats decreased to 0.6 times com-ared with 1-day-old (Fig. 1E). However, the correspond-

ng protein increased with age, being 1.5, 5.1, and 4.9imes higher in 15 day, 2 month and adult cats, respec-ively compared with 1-day-old (Fig. 1F).

Organ-specific ChAT protein expression pattern.hen conducting the experiments reported in Fig. 1B, D

nd F, we observed that 2 min or 30 min of exposure toensitive film was enough to obtain signal for ChAT in theCG or petrosal ganglion, respectively, whereas 2 h was

enzyme in the CB (A and B), petrosal ganglion (C and D), and SCG

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A. Bairam et al. / Neuroscience xx (2007) xxx 5

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ecessary for the CB. This observation suggests thathAT level differs between organs. Hence, other analysesere performed to determine the pattern of expressionithin these tissues specifically. To prevent signal satura-

ion, we used 20 �g of cytosolic protein for the CB, 5 �g forhe petrosal ganglion, and 1 �g for the SCG from 1-day-oldFig. 2A) and adult cats (Fig. 2B), as showed at the top ofach histogram. Signals intensities for the petrosal andCG were first corrected depending on ratio to equal 20 �grotein used for the CB (Fig. 2). The CB contained the

ower ChAT level compared with other organs regard-ess of age. In 1-day-old cat, ChAT level was 1.5 and 28imes higher in the petrosal ganglion and SCG, respec-ively compared with the CB. In adult cats, a similarattern of ChAT expression level between organs wasbserved. However, ChAT protein was 4.4 and 26 timesigher in petrosal and SCG, respectively, compared withhe CB.

Developmental pattern of nicotinic receptor transcriptevels. The relative expression level of mRNA transcriptor �3, �4, �7, and �2 assessed by real-time RT-PCRmplification is shown in Fig. 3.

In the CB, in comparison with 1-day-old cats, theRNA transcript level for the nicotinic �3 and �7 receptors

ncreased particularly in adult, while the nicotinic �4 recep-or decreased gradually with age (Fig. 3A). The mRNAevels for �2 receptors were slightly reduced in 15-day-oldnimals only.

In the petrosal ganglion, in comparison with 1-day-old

ig. 2. ChAT protein profile levels between CB, petrosal ganglion, andre shown at the top of each histogram. Different quantities of total cyere used to prevent saturation allowing evaluation of signal intens

Chemicon); while in the last lane, no primary antibody was used (�c

ats, the mRNA transcript level for the �3 and �4 receptor b

Please cite this article in press as: Bairam A, et al., Developmental proeral chemoreflex pathway in cats, Neuroscience (2007), doi: 10.1016/j.n

ncreased particularly in adults as well as the �2 receptorRNA. However, the �7 receptor mRNA did not changeith age (Fig. 3B).

In the SCG, the pattern of changes of nicotinic receptorRNA levels was similar to that observed in the CB (Fig.C). Compared with 1-day-old cats, the mRNA transcript

evels for the nicotinic �3 and �7 receptors increased sig-ificantly in adult, particularly, for the �7 receptor. Theicotinic �4 receptors mRNA decreased gradually withge, but the �2 receptor mRNA increased modestly in-month-old and adult cats.

We failed to determine the protein level of these nico-inic receptors with commercially available antibodies evenhough some of them were successfully used in immuno-istochemistry technique (Shirahata et al., 1998; Higashit al., 2003).

urinergic system

Developmental pattern of relative ATP content.uciferin-luciferase technique was used to estimate theevelopmental pattern of the relative cytosolic ATP content

n each organ. An example of standard curve for ATPssay is shown as an inset in Fig. 4A.

In the CB, ATP content decreased gradually with ageeing 0.5 times lower in 2-month-old and adult cats than

n 1-day-old (Fig. 4A). In the petrosal ganglion, ATPontent was not affected by age (Fig. 4B). However, inhe SCG, ATP content increased significantly with age

-day-old (A) and adult cats (B). Representative Western blot analysesc protein (20 �g, 5 �g and 1 �g for CB, PG, and SCG, respectively)ween organs. The 1st lane (�ctr) used recombinant ChAT proteinare mean�S.D.; CB, PG (petrosal ganglion), SCG, ctr (control).

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eing about 3.7, 1.8, and 2.7 times higher in 15 day, 2

file of cholinergic and purinergic traits and receptors in periph-euroscience.2007.03.034

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A. Bairam et al. / Neuroscience xx (2007) xxx6

ARTICLE IN PRESS

ig. 3. Relative levels of cholinergic nicotinic receptor transcript in the CB (A), petrosal ganglion (B) and SCG (C) of developing cats as evaluated by

Please cite this article in press as: Bairam A, et al., Developmental profile of cholinergic and purinergic traits and receptors in periph-eral chemoreflex pathway in cats, Neuroscience (2007), doi: 10.1016/j.neuroscience.2007.03.034

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A. Bairam et al. / Neuroscience xx (2007) xxx 7

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onth and adult cats respectively compared with 1-day-ld cats (Fig. 4C).

Developmental pattern for P2X2 mRNA and P2X2 pro-ein expression levels. In carotid bodies from 2 month-ld and adult cats, the P2X2 mRNA levels decreased to 0.5imes the level reported in 1-day-old cats (Fig. 5A), whilerotein levels were not affected by age (Fig. 5B). In theetrosal ganglion, the P2X2 mRNA levels were slightlyffected by age (Fig. 5C), but protein levels were 0.5

imes lower in 2-month-old and adult cats compared with-day-old (Fig. 5D). In the SCG, P2X2 mRNA levelsecreased with age (Fig. 5E). Levels measured in-month-old and adult cats were 0.2 times the levelseported in 1-day-old; however, the protein level was notffected by age (Fig. 5F). Representative Western blotsre shown at the top of histograms B, D and F in Fig. 5.

Developmental pattern for P2X3 mRNA and P2X3 pro-ein expression levels. In the CB, the P2X3 receptorRNA levels decreased gradually with age being 0.5 times

ower in 2-month-old and adult compared with 1-day-oldats (Fig. 6A); however, protein levels were not affected byge (Fig. 6B). In the petrosal ganglion, while the P2X3

eceptor mRNA levels increased with age (Fig. 6C), itsrotein level decreased, being 0.5 times lower in 2-month-ld and adult compared with 1-day-old cats (Fig. 6D). In

he SCG, the P2X3 receptor mRNA levels increased withge (Fig. 6E), whereas protein levels decreased (Fig. 6F).epresentative Western blots are shown at the top ofistogram in Fig. 6 (B, D and F).

Organ-specific pattern of P2X2 and P2X3 protein ex-ression levels. Purinergic receptors were identified aftermin exposure in the petrosal ganglion, compared with

0–45 min for other organs. Hence, as we did for ChATtudies, other analyses were performed to determine tohich extent the expression level of P2X2 and P2X3 re-eptors is organ-dependent. We used 20 �g of membranerotein for the CB, 5 �g for the petrosal ganglion, and

ig. 4. Profile of ATP content in the CB (A), petrosal ganglion (B) andxpressed as relative light units. ATP standard curve is shown as an ins2m).

0 �g for the SCG of 1-day-old and adult cats (Figs. 7 and 2

Please cite this article in press as: Bairam A, et al., Developmental proeral chemoreflex pathway in cats, Neuroscience (2007), doi: 10.1016/j.n

), as showed at the top of each histogram. Signal inten-ities for the petrosal ganglion were first corrected depend-

ng on ratio to equal 20 �g protein used for the CB.For the P2X2 receptor protein (Fig. 7), the petrosal

anglion contained the highest level of P2X2 receptor pro-ein which was 2.5 times higher compared with the CB andCG in either 1-day-old or adult cats (Fig. 7A and B,

espectively).For the P2X3 receptor protein (Fig. 8), again the petro-

al ganglion contained the highest level of this receptor,hich was four times higher compared with CB and SCG inither 1-day-old or adult cats (Fig. 8A and B, respectively).

DISCUSSION

his study describes the postnatal development of cholin-rgic and purinergic systems in CB, petrosal ganglion, andCG, the main component of peripheral chemoreceptorathway. This includes pre- and post-synaptic elementsnd the sympathetic relay that provides motor innervationo the CB in cats. The most important findings are thathAT protein content in CB, petrosal, and superior cervicalanglia increases with advancing age. Moreover, this isccompanied by an augmentation of mRNA expression

evels for �3, �4 and �2 nicotinic receptors subunits in theetrosal ganglion, while in CB and SCG the �4 nicotiniceceptor subunit decreases drastically with age whereashe �3 and �7 expression level increases. Protein levels of2X2 and P2X3 purinergic receptors are not developmen-

ally regulated in carotid bodies, but showed a gradualecline of expression during development in petrosal gan-lion. A similar decrease of protein expression level occurs

n SCG for P2X3 receptors only. Since recent data clearlyemonstrate that hypoxia triggers ATP (Buttigieg andurse, 2004) and ACh release (Fitzgerald et al., 1999;hang et al., 2000; Prabhakar, 2006) from the CB, and thatoth transmitters increase carotid sinus nerve activity inats (Nurse and Zhang, 2001; Rong et al., 2003; Gourine,005) and in cats (Iturriaga et al., in press; Reyes et al.,

of developing cats as evaluated by luciferin-luciferase technique andel A. Data are mean�S.D., 1 day old (1d), 15 days old (15d), 2 months

SCG (C)et in pan

007a,b) (via P2X2 and nicotinic post-synaptic receptors

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A. Bairam et al. / Neuroscience xx (2007) xxx8

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espectively), these results suggest that postnatal devel-pment of nicotinic mechanisms is tightly linked to post-atal development of chemoreceptor functions. Al-hough purinergic mechanisms may also be involved,ur results do not directly support this hypothesis. Fu-ure functional studies should help elucidate these de-elopmental mechanisms.

echnical considerations

lthough we used RNA from whole carotid bodies, we mayikely rule out significant contamination from other struc-ures such as sympathetic or sensory nerve terminals. Asn illustration, ChAT mRNA increases with age in CB andecreases in petrosal or superior cervical ganglia (Fig. 1).ur data did not allow us to determine whether ChATRNA in these organs is present in multiple forms as in theNS of rat (seven) and mice (five) (Wu and Hersh, 1994).lthough all these forms contain the same coding region in

ats or mice (Wu and Hersh, 1994), it is still unknownhether these are differentially regulated during develop-ent. Another observation is the dichotomy betweenRNA and protein levels, which is not uncommon in de-

ig. 5. Developmental pattern of mRNA expression and protein levelsE and F) of developing cats. Representative Western blot analyses fortudied. Data are mean�S.D., 1 day old (1d), 15 days old (15d), 2 m

elopmental studies (Bairam et al., 2006; Rage et al., c

Please cite this article in press as: Bairam A, et al., Developmental proeral chemoreflex pathway in cats, Neuroscience (2007), doi: 10.1016/j.n

006). For ChAT this dichotomy is related to specific-egion-regulation of ChAT mRNA and gene transcriptionWu and Hersh, 1994).

holinergic system

ur results showing the presence of both ChAT mRNAnd protein in the CB of adults and newborn cats areonsistent with previous immunohistological demonstra-ion of ChAT protein expression in CB type I cells in adultats (Wang et al., 1989). A large amount of data currentlyupports the ability of chemoreceptor cells to synthesizend release ACh upon hypoxic exposure (Fitzgerald et al.,999). Clusters of type I cell from 7- to 14-day-old rats are

mmunopositive for vesicular ACh transporter (Nurse andhang, 1999, 2001; Zhang and Nurse, 2004); while Wangt al. (1989) used immunohistochemistry to show that theajority of type I cells of adult cat and rabbit contain ChAT.

n similar lines of evidence, Kim et al. (2004) showed thatCh itself is present in many primary cultured CB type Iells from rabbits where it co-localized with tyrosine hy-roxylase. From a functional point of view, electrophysio-

ogical studies have consistently shown that ACh acts

2 receptor in the CB (A and B), petrosal ganglion (C and D) and SCGeptor are shown at the top of histograms B, D, and F for each of organs

).

for P2X

oordinately with ATP and is critically involved in neural

file of cholinergic and purinergic traits and receptors in periph-euroscience.2007.03.034

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A. Bairam et al. / Neuroscience xx (2007) xxx 9

ARTICLE IN PRESS

ransmission between CB type I cells and sensitive end-ngs of the carotid sinus nerve (Zhang et al., 2000; Rong etl., 2003; Varas et al., 2003). Nonetheless, a recent studysing in situ hybridization and immunohistochemistry failedo detect ChAT or the vesicular ACh transporter in the CBf newborn or post-weanling rats (28-day-old) (Gauda etl., 2004). Some technical aspects concerning this partic-lar study have been discussed in detail elsewhere (Shira-ata et al., 2007). Nonetheless, some functional studiesave not been able to report a key role for ACh in CBhemotransduction (Spergel and Lahiri, 1993; Reyes et al.,007a), which still clearly questions the cholinergic hypoth-sis. A complementary hypothesis proposes that adeno-ine, whether released from chemosensitive cells or gen-rated by the breakdown of ATP within the synaptic cleft,ontributes to the generation of the postsynaptic actionotential (McQueen and Ribeiro, 1981, 1986), and it haseen recently shown that endogenous adenosine contrib-tes to the carotid sinus nerve response to hypoxia actingoth on pre- and postsynaptic adenosine receptors (Condet al., 2006). Interestingly, activation of �4 nicotinic recep-or in rat CB also increases adenosine release, which mayct as a positive feedback loop to further increase CNS

ig. 6. Developmental pattern of mRNA expression and protein levelsE and F) of developing cats. Representative Western blot analyses fortudied. Data are mean�S.D., 1 day old (1d), 15 days old (15d), 2 m

esponse (Conde and Monteiro, 2006). 2

Please cite this article in press as: Bairam A, et al., Developmental proeral chemoreflex pathway in cats, Neuroscience (2007), doi: 10.1016/j.n

On this background, our results show that ChAT pro-ein levels increase during postnatal development in cat’sB, petrosal, and superior cervical ganglia (Fig. 1). Al-

hough the magnitude of this increase is less in the CBompared with other organs, it suggests that ACh synthe-is is age dependent at the level of sensory organs (CBnd petrosal ganglion) as well as at the level of sympa-hetic organ (SCG).

At all ages investigated, the ChAT protein level wasbout 25–30 times lower in CB than in SCG. Hence, ques-ions concerning the significance of ACh content in the CBay arise. In the SCG of adult cats, ACh content is aroundnmol/mg protein, with a specific ChAT activity of 50 nmol//mg protein (Tandon et al., 1996). By contrast, the contentf ACh in adult cat’s CB has been found to be around0 nmol/g tissue (Fidone et al., 1976) i.e. about 50 times

ower than in SCG. With a content of ChAT being 25 timesower in the CB compared with SCG (as reported here),pecific ChAT activity would be 2 nmol/h/mg protein if onessume similar regulation between the CB (CB) and SCG.his estimated value of ACh turnover is close to whateported in cholinergic areas of the CNS in which AChurnover is around 0.5–1 nmol/h/mg protein (Smith et al.,

3 receptor in the CB (A and B), petrosal ganglion (C and D) and SCGeptor are shown at the top of histograms B, D, and F for each of organs

).

for P2X

004), this seems to be sufficient to support a role for ACh

file of cholinergic and purinergic traits and receptors in periph-euroscience.2007.03.034

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A. Bairam et al. / Neuroscience xx (2007) xxx10

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n carotid sinus nerve activity and response to hypoxia. Onhe other hand, in steady-state conditions, a low level of

ig. 7. P2X2 protein receptor profile levels between CB, petrosal gangnalyses were shown at the top of each histogram. Different quantitiespectively) were used to prevent saturation, thus allowing evaluationhe inner one the primary antibody was pre-incubated with its specific

ig. 8. P2X3 protein receptor profile levels between CB, petrosal gangnalyses were shown at the top of each histogram. Different quantiti

Please cite this article in press as: Bairam A, et al., Developmental proeral chemoreflex pathway in cats, Neuroscience (2007), doi: 10.1016/j.n

Ch content in CB may account for high turnover rate. Thisay be the case even with low level of ChAT, since ChAT

SCG in 1-day-old (A) and adult cats (B). Representative Western blotl membrane protein (20 �g, 5 �g and 20 �g for CB, PG, and SCG,

l intensities between organs. The last two lanes are controls (�ctr). In(Alomone), while in the last one no primary antibody was used.

SCG in 1-day-old (A) and adult cats (B). Representative Western blotl membrane protein (20 �g, 5 �g and 20 �g for CB, PG, and SCG,

lion andes of tota

lion andes of tota

l intensities between organs. The last two lanes are controls (�ctr). In(Alomone), while in the last one no primary antibody was used.

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A. Bairam et al. / Neuroscience xx (2007) xxx 11

ARTICLE IN PRESS

ctivity may not be directly related to its mRNA or proteinevel (Wu and Hersh, 1994; Berrard et al., 1986). Further-

ore, a splice-variant form of ChAT has been reported ineripheral nervous system (Chiocchetti et al., 2003;imura et al., 2007; Yasuhara et al., 2007), but its expres-ion has not been tested in the CB. As a conclusion, untilefinitive data on expression of peripheral ChAT isoformnd ACh turnover rate have been reported in the CB,omparison with the SCG for the specific significance ofhAT protein level as reported in the present study re-ains highly speculative.

We also report the postnatal development of mRNAxpression for nicotinic receptor subunits �3, �4, �7 and �2.hile such developmental data were previously not avail-

ble for CB and petrosal ganglion in any species, ourctual results for SCG are similar to those observed in ratsMandelzys et al., 1994) where the mRNA for �3 and �7

icotinic receptors genes expression increased abouthreefold over the first 2 weeks of life.

Physiological significance. The gradual increase ofhAT protein during development in CB and petrosal gan-lion (sensory elements) suggests that ACh synthesis in-reases with age. This developmental pattern of ChATrotein strongly contrasts with tyrosine hydroxylase ex-ression, the enzyme responsible of dopamine synthesis,

n cats (Bairam et al., 2006). Tyrosine hydroxylase mRNAnd protein decrease significantly with age, while expres-ion of D2 dopamine receptors increases with age, as wells the functional dopaminergic inhibition of intracellularalcium from rat cultured type I cells (Bairam and Carroll,005; Carroll et al., 2005; Bairam et al., 2006). It seemseasonable to hypothesize that interactions between cho-inergic and dopaminergic influences on CB chemosensoryctivity are developmentally regulated. In adult cats andabbits, ACh and dopamine regulate their own releaserom the CB (Obeso et al., 1997; Bairam et al., 2000; Wangnd Fitzgerald, 2002; Kim et al., 2004). The age dependent

ncrease of ChAT protein in sensory elements, indirectvidence of an increase in ACh level, suggests a betterxpression of ACh excitatory role on chemosensory activ-

ty with age and, in parallel, its role to counteract thenhibitory effect of dopamine, which was previously re-orted in adult cats (Bairam and Lajeunesse, 2003; Bairamnd Marchal, 2003).

Concerning post-synaptic elements (i.e. cholinergic re-eptors), although, the reported changes in mRNA expres-ion levels for nicotinic receptors cannot predict similarhanges in final protein, they at least suggest that nicotiniceceptors genes’ expression is developmentally regulated.ach of these nicotinic receptors may have a determinedhysiological role, which may change during development.s an example, besides the key role of nicotinic receptorsn carotid sinus nerve response to hypoxia in adults rats,ice or cats (Zhang et al., 2000; Rong et al., 2003; Varast al., 2003; Zhang and Nurse, 2004; Alcayaga et al.,007), activation of �4 and �2 subunits in CB by AChroduces an increase in extracellular adenosine level

Please cite this article in press as: Bairam A, et al., Developmental proeral chemoreflex pathway in cats, Neuroscience (2007), doi: 10.1016/j.n

ffects exist in newborns. Concerning the SCG, activationf nicotinic cholinergic receptors is the major way of syn-ptic transmission (Alkadhi et al., 2005). The clear dra-atic increase in ChAT level and the changes in the profilef nicotinic receptors, at least for �4 and �7, with age

ndicate that sympathetic influence on CB function throughnternal and external ganglionic carotid nerve is age-de-endent in cats.

urinergic system

ATP assay. Luciferin-luciferase technique was previ-usly used to estimate the effect of hypoxia on CB ATPontent in adult cats (Acker and Starlinger, 1984; Obeso etl., 1985) or rabbits (Verna et al., 1990), however, theesults were not conclusive. It is nonetheless clearly es-ablished that hypoxia increases ATP release from rat CBn vitro (Buttigieg and Nurse, 2004). However, the devel-pmental aspect of ATP content in chemosensory organsas not been established previously. Under our experi-ental conditions, ATP content decreases during devel-pment in the CB, increases in the SCG, and does nothange in the petrosal ganglion (Fig. 5). While, they clearlyave an intrinsic value, these results are difficult to inter-ret and put into perspective to the consider potential

mplication of ATP on postnatal development of chemo-ensory functions. ATP is widely used in the cells as a highnergy–carrying molecule, and the fraction of ATP de-oted to neural transmission should be negligible. At theest, we may speculate that ATP level in each organ iselated to particular metabolic needs. Since CB functionnd oxygen sensing mechanisms mature progressivelyfter birth (Carroll, 2003; Donnelly, 2005) it is tempting topeculate that this postnatal development requires aigher amount of energy than the maintenance of CBunction in adults.

Purinergic receptors. Three interesting findings cane inferred from our study. First: there is a similar devel-pmental profile of protein and mRNA for P2X2 (Fig. 5) and2X3 (Fig. 6) in CB and petrosal ganglion. Second: theighest protein level of P2X2 and P2X3 is found in theetrosal ganglion vs. CB and SCG. Third: only P2X3 butot P2X2 receptor protein decreased with age in the SCG,

n line with what was observed in rats (Dunn et al., 2005),here the functional expression of P2X3 receptor (wholeell patch clamp) and its density (immunohistochemistry)ecrease during development (Dunn et al., 2005). A pre-ominant role was proposed for P2X3 receptors in theynaptogenesis process during early days of life (Dunn etl., 2005).

hysiological significance

n central and peripheral nervous system ATP is co-storedn synaptic vesicles containing classical transmitters. Inholinergic vesicles, the ratio of ACh/ATP varies between:1–10:1 (Zimmermann, 1994), and ATP co-released withCh acts as transmitter or co-transmitter to increaseostsynaptic neuronal activities (Bodin and Burnstock,

file of cholinergic and purinergic traits and receptors in periph-euroscience.2007.03.034

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A. Bairam et al. / Neuroscience xx (2007) xxx12

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ablished that hypoxia increases ATP release from chemo-ensitive cells and that activation of P2X2 receptorsauses depolarization of chemosensitive nerve endingssee for recent review Zapata, 2007). Overall, the excita-ory effect of ATP on carotid sinus nerve activity is about0-times lower than nicotine (Reyes et al., 2007b). Be-ause protein level of P2X2 receptors decreases duringevelopment in petrosal ganglion, it seems rather hazard-us to hypothesize an implication of purinergic signaling onhe postnatal development of peripheral chemoreceptorunction, unless the observed decline in P2X2 receptorxpression reflects a higher receptor turnover triggered bynhanced purinergic signaling. However, this would noteflect a developmental up-regulation of postsynaptic pu-inergic receptor accompanying the development of che-osensory responses.

CONCLUSION

n conclusion, our study showed that, in 1-day-old cats, theeural elements that are part of the chemosensory afferentathway contain a low level of ChAT enzyme comparedith adults, thereby suggesting (albeit indirectly) that ACh

evels in newborns are lower than in adult animals. Con-equently, we propose that the low chemosensory activityn response to hypoxia in newborn animals is related (ateast in part) to low ACh levels. Our findings stronglyuggest that in cats, the age-related changes in cholinergicystem in peripheral chemosensory elements play a de-erminant role in the postnatal development of chemore-eptor function. While our results do not provide directvidence supporting a key role for the purinergic systemuring this postnatal development, this hypothesis war-ants further studies before it is dismissed.

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(Accepted 23 March 2007)

file of cholinergic and purinergic traits and receptors in periph-euroscience.2007.03.034