Yves Lajeunesse

Neonatal maternal separation and early life programming of the hypoxic ventilatory response in rats.

The neonatal period is critical for central nervous system (CNS) development. Recent studies have shown that this basic neurobiological principle also applies to the neural circuits regulating respiratory activity as exposure to excessive or insufficient chemosensory stimuli during early life can have long-lasting consequences on the performance of this vital system. Although the tactile, olfactory, and auditory stimuli that the mother provides to her offspring during the neonatal period are not directly relevant to respiratory homeostasis, they likely contribute to respiratory control development. This review outlines the rationale for the link between maternal stimuli and programming of the hypoxic ventilatory response during early life, and presents recent results obtained in rats indicating that experimental disruption of mother-pup interaction during this critical period elicits significant phenotypic plasticity of the hypoxic ventilatory response.

Developmental profile of cholinergic and purinergic traits and receptors in peripheral chemoreflex pathway in cats

This study describes the developmental profile of specific aspects of cholinergic and purinergic neurotransmission in key organs of the peripheral chemoreflex: the carotid body (CB), petrosal ganglion (PG) and superior cervical ganglion (SCG). Using real time RT-PCR and Western blot analyses, we assessed both mRNA and protein expression levels for choline-acetyl-transferase (ChAT), nicotinic receptor (subunits alpha3, alpha4, alpha7, and beta2), ATP and purinergic receptors (P2X2 and P2X3). These analyses were performed on tissue from 1- and 15-day-old, 2-month-old, and adult cats. During development, ChAT protein expression level increased slightly in CB; however, this increase was more important in PG and SCG. In CB, mRNA level for alpha4 nicotinic receptor subunit decreased during development (90% higher in 1-day-old cats than in adults). In the PG, mRNA level for beta2 nicotinic receptor subunit increased during development (80% higher in adults than in 1-day-old cats). In SCG, mRNA for alpha7 nicotinic receptor levels increased (400% higher in adults vs. 1-day-old cats). Conversely, P2X2 receptor protein level was not altered during development in CB and decreased slightly in PG; a similar pattern was observed for the P2X3 receptor. Our findings suggest that in cats, age-related changes in cholinergic and purinergic systems (such as physiological expression of receptor function) are significant within the afferent chemoreceptor pathway and likely contribute to the temporal changes of O2-chemosensitivity during development.

Expression of sex-steroid receptors and steroidogenic enzymes in the carotid body of adult and newborn male rats.

This study describes the localization and pattern of expression of estradiol and progesterone receptors as well as key enzymes for steroid synthesis (i.e. P450 side-chain-cleavage--P450scc, and P450 aromatase--P450Aro) in the carotid body (CB) and superior cervical ganglion (SCG) of adult, newborn and late fetal male rats, using immunohistochemistry, Western blot and real-time RT-PCR. Our results show a constitutive expression of the beta estradiol receptor (Erbeta) and the 80 kDa and 60 kDa progesterone receptors (PR-A and PR-C) isoforms in the CB, while in the SCG Eralpha, Erbeta, PR-A and PR-C are expressed. While P450Aro staining was negative, P450scc staining was strong both in the SCG and CB. In late fetal and newborn rats, Eralpha was not detected in the CB or SCG, but a slight staining appeared for P450 aromatase in the CB, and to a lesser extent in SCG. P450scc was strongly expressed in CB and SCG of late fetal and newborn rats. We conclude that the carotid body shows a constitutive expression of Erbeta and PR and may be able to synthesize steroids, including estradiol during late fetal life.

Developmental pattern of M1 and M2 muscarinic gene expression and receptor levels in cat carotid body, petrosal and superior cervical ganglion

Using real-time reverse transcriptase polymerase chain reaction, Northern blot, and Western blot analyses, we evaluated the developmental pattern of mRNA and protein expression level of muscarinic M1 and M2 receptors in the carotid body, petrosal ganglion and superior cervical ganglion of 1-day, 15-day, 2-month-old and adult cats. mRNA expression and protein levels of tyrosine hydroxylase, the rate limiting enzyme for dopamine synthesis, were also assessed. Carotid body M1 receptor mRNA, increased significantly by approximately 100% and 300% in 2-month and adult vs. 1- and 15-day-old cats, but protein level decreased gradually being approximately 50% lower compared with 1-day-old cats. In the petrosal ganglion, muscarinic M1 receptor mRNA level was higher in 15-day-old cats vs. 1-day-old, 2-month-old and adult cats and protein levels were about 30% lower than in 1- and 15-day-old cats. In the superior cervical ganglion, muscarinic M1 receptor mRNA was approximately 50% and 80% higher in 2-month-old and adult cats than 1- and 15-day-old, but no changes in the protein level except in 15-day-old cats which was approximately 40% higher than 1-day-old. There was no change of muscarinic M2 receptor mRNA or protein level in the carotid body or petrosal ganglion. However, in the superior cervical ganglion, the significant increase of mRNA of 30% and 50% in 2-month-olds and adults, respectively was not associated with an increase in receptor protein. Tyrosine hydroxylase mRNA and protein level decreased significantly with age in the carotid body and petrosal ganglion. In the superior cervical ganglion, the age dependent increase in tyrosine hydroxylase mRNA was not associated with any changes in the protein level. These results show that the expression of muscarinic M1 and M2 receptors are age and organ-dependent in cats. Consequently, these changes may modulate chemosensory activity during development since muscarinic M1 receptor is predominantly involved in postsynaptic chemosensory activity, while muscarinic M2 receptor modulates acetylcholine and dopamine release from chemosensitive cells.

Altered expression of adenosine A1 and A2A receptors in the carotid body and nucleus tractus solitarius of adult male and female rats following neonatal caffeine treatment

Neonatal caffeine treatment (adenosine receptor antagonist, 15 mg/kg/day, between postnatal days 3 and 12) affects respiratory patterns in adult male but not female rats as shown by an increase in the respiratory frequency in the early phase of response to hypoxia and an increase in the tidal volume in the late phase of response. Here, we tested the hypothesis that these changes are correlated with modified expression of adenosine receptors in the chemoreflex pathway. Carotid bodies, nucleus tractus solitarii, and superior cervical ganglia were collected from 3-month-old male and female rats that were either naive (not manipulated during the neonatal period) or treated with caffeine (NCT) or water (NWT) between postnatal days 3 and 12 by gavage. Western blot analysis was used to assess the expression of adenosine A(1) and A(2A) receptors and tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis. In male rats, there was a 37% increase in the level of A(2A) receptor and a 17% decrease in tyrosine hydroxylase in the carotid body of NCT (p<0.001) as compared to NWT rats. In the nucleus tractus solitarius, we found a 13% and 19% decrease in A(1) receptor expression in NWT and NCT rats (p<0.01), respectively, compared to naive rats. In the superior cervical ganglion, there was no change in A(1) receptor, A(2A) receptor, and tyrosine hydroxylase expression. In female rats, the only changes observed were decreases of 12% and 15% in A(1) receptor levels in the nucleus tractus solitarius of NWT and NCT rats (p<0.01), respectively, compared to naive rats. We conclude that NCT induces long-term changes in the adenosine receptor system. These changes may partially explain the modifications of the respiratory pattern induced by NCT in adults. The increased expression of the adenosine A(2A) receptor (specific to male rats), combined with the decreased tyrosine hydroxylase expression in the carotid body, suggests that NCT affects adenosine-dopamine interactions regulating chemosensory activity.

Enhancement of the breathing frequency response to hypoxia by neonatal caffeine treatment in adult male rats: The role of testosterone

Caffeine is a common treatment for apnea of prematurity. Although relatively safe, little is known about the potential long-term effects of this treatment on respiratory control development. We previously showed that adult male (but not female) rats previously subjected to neonatal caffeine treatment (NCT; 15 mg/kg/day, postnatal days 3-12) show a higher breathing frequency response during the early phase of hypoxic exposure. To address the role of sexual hormones in this sexual dimorphism, the present study tested the hypothesis that in adult male rats, circulating testosterone contributes to NCT-related augmentation of the acute breathing frequency response to hypoxia. Whole body plethysmography was used to compare the acute ventilatory response to moderate hypoxia (FIO2=0.12; 20 min) between rats previously subjected to NCT or neonatal water treatment (NWT; same treatment as NCT but using water). In each group, rats were either sham-operated or gonadectomized (GDX) 14 days prior to ventilatory measurements. In sham-operated rats, the increase in breathing frequency measured during the first 8 min of hypoxia was greater in NCT rats versus NWT. The hypoxic ventilatory response measured at the end of the hypoxia was not affected by treatment, thus indicating that NCT mainly affected the peripheral component of the chemoreflex. Gonadectomy had no effect on NCT but augmented the frequency response of NWT rats to the same level of NCT, thus eliminating the between-group difference. NCT may interfere with the inhibitory effect of circulating testosterone on carotid body function. Although appealing, additional experiments are necessary to substantiate this interpretation.

Neonatal caffeine treatment does not induce long-term consequences on TrkB receptors or BDNF expression in chemosensory organs of adult rats.

Chronic treatment with caffeine during the neonatal period (neonatal caffeine treatment, NCT, 15mg/kg/day from P3 to P12, oral gavage) has long-lasting consequences on respiratory control development. In adult male (but not female) rats, prior exposure to NCT results in a greater respiratory frequency response to hypoxia. This sex-specific effect of NCT was accompanied by an augmented expression of adenosine A(2A) receptors (A(2A)R) in the carotid body (CB) but not in the nucleus tractus solitarius (NTS). Since activation of adenosine A(2A)R can directly stimulate synthesis of tyrosine kinase B receptor (TrkBR) and brain-derived neurotrophic factor (BDNF), we determined whether NCT increases TrkBR and BDNF expression levels in the CB and NTS using both RT-PCR and western blot analyses. CB, NTS, and superior cervical ganglion were collected from adult male and female rats (10-12 weeks old) previously subjected to NCT or to control (neonatal water treatment, NWT). In male rats, when NCT tended to decrease TrkBR mRNA transcript levels by about 32% in the CB and to reduce BDNF transcripts in the NTS by 22%, western blot analyses showed no parallel changes in final protein expression. NCT had no effects on TrkBR or BDNF mRNA and protein levels in the CB and NTS of female rats. Neither gene was altered by NCT in the superior cervical ganglion of male and female rats. These data suggest that NCT has no long-term effects on trophic factor BDNF and TrkBR expression at peripheral and central level of chemosensory organs involved in respiratory control.

Time Dependent Regulation of Dopamine D1- and D2- Receptor Gene Expression in the Carotid Body of Developing Rabbits by Hypoxia

In the central nervous system, activation of dopamine (DA) receptors (R) by their substrate, DA, modulates the regulation of genes encoding DA Rs themselves (Sidhu et al., 1999; Missale et al., 1998; Gerfen et al., 1990). These effects were shown to be: 1) tissue and region specific, 2) dependent on physiological function of activated DA Rs, and, 3) due to the synergistic effect of stimulation of DA D1 and D2 Rs types (Missale et al., 1998; Gerfen et al., 1990).

Concomitant Effect of Acetylcholine and Dopamine on Carotid Chemosensory Activity in Catecholamine Depleted Cats

Carotid body chemotransduction mechanisms involve many neurotransmitters that are synthetized, stored, and released from the chemoreceptor cell. Acetylcholine (ACh) and dopamine (DA) are the main transmitters studied to date. It has been suggested that, at least in cats, ACh is an excitatory whereas DA is an inhibitory transmitter in the carotid body (as review see Eyzaguirre and Zapata, 1884; Fidone et al., 1997; Fitzgerald, 2000). However, their role on chemosensory activity in response to hypoxia is still debated. Recently, in catecholamine-depleted cats where the storage and release of carotid body DA was greatly impeded by the use of ∝-methyl-paratyrosine and reserpine (Bairam and Marchai, in press), DA infiision was showed to inhibit the carotid sinus nerve chemosensory discharge rate (CSND) while ACh reversed the effect under basal condition. In response to hypoxia, DA slowed the initial increase of CSND whereas ACh accelerated it, while neither drug altered the steady-state chemosensory discharge under hypoxic conditions. One interpretation of these results was that DA infusion prevented the expression of ACh excitatory effect during hypoxia as it was infused methyl-paratyrosine maintained throughout ACh administration. Using a similar model of adult cats pre-treated with oc-methyl-paratyrosine and reserpine (Bairam and Marchai, in press), we investigated the effect of 1) ACh infusion on CSND response to different inspiratory oxygen concentrations (Fi02) and 2) DA infusion while ACh is maintained. This protocol of drug administration should reveal the excitatory role of ACh on CSND under basal condition particularly in hypoxia. Indeed, the methyl-paratyrosine with oc-methyl-paratyrosine and reserpine, which inhibits and depletes catecholamine synthesis and storage (Leitner and Roumy, 1986; Fitzgerald et al., 1983), should minimize the effects of endogenous release of DA on CSND.