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.

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 maternal separation disrupts regulation of sleep and breathing in adult male rats.

STUDY OBJECTIVES: Neonatal maternal separation (NMS) disrupts development of cardiorespiratory regulation. Adult male rats previously subjected to NMS are hypertensive and show a hypoxic ventilatory response greater than that of controls. These results have been obtained in awake or anesthetised animals, and the consequences of NMS on respiratory control during normal sleep are unknown. This study tested the following. HYPOTHESES: NMS augments respiratory variability across sleep-wake states, and NMS-related enhancement of the hypoxic ventilatory response occurs during sleep. METHODS: Two groups of adult rats were used: controls (no treatment) and rats subjected to NMS. Ventilatory activity, coefficient of variation, and hypoxic ventilatory response were compared between groups and across sleep-wake states. SUBJECTS: Male Sprague Dawley rats-NMS: n=11; controls: n=10. Pups subjected to NMS were isolated from their mother for 3 hours per day from postnatal days 3 to 12. Controls were undisturbed. MEASUREMENTS AND RESULTS: At adulthood, sleep-wake states were monitored by telemetry, and ventilatory activity was measured using whole-body plethysmography. Sleep and breathing were measured for 2.5 hours (in the morning) while the rats were breathing room air. Data were analysed in 20-second epochs. Rats were then exposed to a brief (90-sec) hypoxic episode (nadir = 12% O2) to measure the hypoxic ventilatory response. The coefficient of variability for tidal volume and breathing frequency decreased during sleep but remained more elevated in NMS rats than in controls. During non-rapid eye movement sleep, the breathing-frequency response to hypoxia of NMS rats was significantly greater than that of controls. CONCLUSION: Neonatal maternal separation results in persistent disruption of respiratory control during sleep.

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.

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.

Neonatal maternal separation enhances dopamine D(2)-receptor and tyrosine hydroxylase mRNA expression levels in carotid body of rats.

Adult male (but not female) rats previously subjected to neonatal maternal separation (NMS) are hypertensive and show a significant increase (25%) in their hypoxic ventilatory response. To begin investigating the mechanisms involved in this gender-specific disruption in cardiorespiratory regulation, we tested the hypothesis that NMS alters the expression of dopamine D(2)-receptors and tyrosine hydroxylase mRNA in 3 peripheral organs involved in cardio respiratory regulation: the carotid bodies, superior cervical ganglia, and adrenals. Pups subjected to NMS were placed in a temperature- and humidity-controlled incubator 3 h per day for 10 consecutive days (P3-P12). Control pups were undisturbed. Once they reached adulthood (8-10 weeks), male and female rats were anesthetised. The carotid bodies, superior cervical ganglia, and adrenals were harvested for semi-quantitative analyses of dopamine D(2)-receptors and tyrosine hydroxylase mRNA expression using reverse transcription-polymerase chain reaction (carotid bodies only) and Northern blot. In the carotid bodies, comparison of densitometric analyses showed that NMS enhanced tyrosine hydroxylase mRNA expression in male, but not female, rats. Neonatal maternal separation increased dopamine D(2)-receptor mRNA expression also, but the effect was not gender specific. No changes in mRNA expression related to dopaminergic neurotransmission were observed in superior cervical ganglia or the adrenals. These results indicate that subsequent mechanistic investigations should focus on the carotid bodies, as enhancement of dopaminergic neurotransmission within this organ likely contributes to the gender-specific effects of NMS on cardiorespiratory regulation.