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1 Pediatrics Research Unit, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Université Laval, Québec City, QC, Canada 2 Neuroscience Research Unit, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Université Laval, Québec City, QC, Canada
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Abstract |
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. Measurement of c-fos mRNA expression in the paraventricular nucleus of the hypothalamus (PVH) combined with plasma ACTH and corticosterone levels confirmed that NMS effectively disrupted HPA axis function in males. In males, baseline minute ventilation was not affected by NMS. In contrast, NMS females show a greater resting minute ventilation due to a larger tidal volume. The hypoxic ventilatory response of male NMS rats was 25% greater than controls, owing mainly to an increase in tidal volume response. This augmentation of the hypoxic ventilatory response was sex-specific also because NMS females show an attenuated minute ventilation increase. Baseline mean arterial blood pressure of male NMS rats was 20% higher than controls. NMS-related hypertension was not significant in females. The mechanisms underlying sex-specific disruption of cardio-respiratory control in NMS rats are unknown but may be a consequence of the neuroendocrine disruption associated with NMS. These data indicate that exposure to a non-respiratory stress during early life elicits significant plasticity of these homeostatic functions which persists until adulthood.
(Received 6 August 2003;
accepted after revision 17 November 2003;
first published online 21 November 2003)
Corresponding author R. Kinkead: Centre de Recherche (D0-711), Hôpital St-François d'Assise, 10 rue de l'Espinay, Québec, QC, G1L 3L5, Canada. Email: richard.kinkead{at}crsfa.ulaval.ca
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Introduction |
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Much like the respiratory control system, ‘programming’ of the hypothalamic–pituitary–adrenal (HPA) axis, the effector of the neuroendocrine response to stress (Herman & Cullinan, 1997; Francis et al. 1999), is also sensitive to early life experiences. The neonatal period from days 3–14 has been described as a ‘stress hyporesponsive period’ that is critical for the maturation of the HPA axis (Wigger & Neumann, 1999). During the neonatal period, mothers provide tactile and chemical stimuli to their offspring that are necessary to proper pups' HPA development and function. Consequently, acute or repeated long-term separation from the dam is one of the most potent naturally occurring stressors to which rat pups can be exposed during the ‘stress hyporesponsive period’ (Wigger & Neumann, 1999). Maternal separation not only deprives the pups of maternal stimulation during the procedure, but also reduces and deranges maternal behaviour upon reunion with the pups (Ladd et al. 2000; Sanchez et al. 2001). As a result of this adverse early life experience, male rat pups subjected to neonatal maternal separation (NMS) are characterized by enhanced behavioural and neuroendocrine responses to stress in adulthood, as they show greater plasma adrenocorticotropic hormone (ACTH) and corticosterone in response to acute stress (for review, see Francis et al. 1999). This hyper-responsiveness to stress, relative to undisturbed (control) rats, is mainly due to a disruption of the paraventricular nucleus of the hypothalamus (PVH) glucocorticoid autoreceptor system which inhibits corticotropin releasing factor (CRF) synthesis and release (Francis & Meaney, 1999).
Although the PVH is a key component of the HPA axis, there is growing evidence indicating that this nucleus is also involved in respiratory regulation. Neuroanatomical tracing studies have shown that PVH neurones send direct projections onto phrenic (inspiratory) motoneurones (Yeh et al. 1997; Kc et al. 2002; Mack et al. 2002), and chemical PVH activation or disinhibition increases diaphragmatic EMG in anaesthetized rats (Yeh et al. 1997; Schlenker et al. 2001). Moreover, prolonged (3 h) exposure to moderate hypoxia 
increases Fos immunolabelling within the PVH, thereby suggesting that activation of PVH neurones may modulate respiratory chemoreflexes (Berquin et al. 2000a, b).
In light of these data suggesting both anatomical and functional overlap between neuroendocrine stress response and ventilatory control, it is conceivable that early life exposure to a non-respiratory stressor, such as NMS, alters development of the respiratory control system. To test this hypothesis, we used whole body plethysmography to compare the hypoxic ventilatory response of adult awake rats previously subjected to NMS to that of undisturbed (control) animals. Given that the effects of NMS are more pronounced in males than in females (Kuhn & Schanberg, 1998; Wigger & Neumann, 1999; Matthews et al. 2001), these experiments were performed on animals of both sexes to determine whether the effects of NMS on the hypoxic ventilatory response development are sex-specific.
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Methods |
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Experiments were performed on 75 male and 51 female Sprague–Dawley rats (Charles River Canada, St Constant, Québec, Canada). Rats were supplied with food and water ad libitum and maintained in standard laboratory conditions (21°C, 12 : 12 h dark–light cycle: lights on at 06.00 h and off at 18.00 h). Laval University Animal Care Committee approved the experimental procedures described in this manuscript, and the protocols were in accordance with the guidelines detailed by the Canadian Council on Animal Care.
Mating and neonatal maternal separation (NMS) procedures
Virgin females were mated and delivered 10–15 pups. Two days after delivery, litters were culled to 12 pups, when necessary, with a roughly equal number of males and females. The NMS protocol was inspired by that of Wigger & Neumann (1999). Briefly, the entire litter was separated daily from their mother for 3 h per day (09.00–12.00 h) from days 3–12. Separated pups were placed in a temperature (35°C) and humidity (45%) controlled incubator and isolated from each other by a cardboard partition. On day 21, rats were weaned and housed under standard animal care conditions until adulthood (8–10 weeks old; see Table 1 for between-group comparison of age and weight data), at which time ventilatory and cardiovascular measurements were performed. Brains were harvested and blood was collected for plasma hormone level measurements in some experiments. The ventilatory, cardiovascular, and neuroanatomical data obtained from this experimental group were then compared to those of animals not subjected to the NMS procedure and continuously maintained under standard animal care procedures. These animals are the most desirable control group for investigations of the effects of maternal separation (Lehmann & Feldon, 2000).
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Surgical procedures
In 37 animals, a catheter was placed in the femoral artery for measurement of arterial blood gases, mean arterial blood pressure, and heart rate according to our standard protocol (Kinkead et al. 2001). Briefly, surgery was performed on NMS (7 males, 5 females) and control (15 males, 10 females) rats under isoflurane anaesthesia (2–2.5% in air). Once in position, the catheter was routed under the skin to the back of the neck and filled with heparinized saline (10 U ml-1). Post-surgery care consisted of three subcutaneous injection of a non-steroid anti-inflammatory (ketoprophene; 2 mg kg-1) immediately after the surgery, 24 and 48 h post-op. The catheter was flushed once daily with heparinized saline to ensure patency. Rats recovered at least 72 h before cardiovascular and ventilatory measurements were made. We compared ventilatory variables of operated and non-operated rats. The surgical procedure had no effect on minute ventilation (P = 0.12), respiratory frequency (P = 0.1), tidal volume (P = 0.77) and CO2 production (P = 0.81). In the absence of statistically significant differences, group-specific data from operated and non-operated rats were pooled for subsequent analysis.
Ventilation and arterial blood gas measurements
Measurements of minute ventilation 
, breathing frequency (f), and tidal volume (VT) in unrestrained rats were obtained by whole body flow-through plethysmograph (model PLY3223, Buxco Electronics, Sharon, CT, USA) according to our method previously described (Kinkead et al. 2001). Briefly, the system consisted of a 4.5 l Plexiglas experimental chamber. The flow of air or hypoxic gas mixture delivered to the chamber was kept constant and ranged between 2.0 and 2.5 l min-1. Rectal temperature was measured before and after each experiment. Barometric pressure, chamber temperature and humidity were also measured to express VT in millilitres (BTPS) per 100 g. The gas mixture flowing out of the chamber was analysed with a flow-through capnograph (Novametrix, Wallingford, CT, USA) for subsequent calculation of CO2 production 
with an open system (Mortola & Dotta, 1992).
The rat was placed in the chamber with room air flowing through. When necessary, the arterial catheter was connected to the swivel for blood sampling and measurements of cardiovascular variables (see below). The rat was allowed to acclimatize to the chamber for roughly 1 h, and baseline (normoxic) measurements were made when the animal was quiet but awake, and the ventilatory and cardio-vascular variables were regular. Experience showed us that with this protocol, the coefficient of variation for breathing frequency and tidal volume over the 10-min baseline period ranges between 10 and 12%; for heart rate and blood pressure, the coefficient of variation was less that 10%. The baseline values obtained were representative of the data recorded over the preceding 30–45 min. In operated rats, a first arterial blood sample of 100 µl was taken at that time. Blood samples were analysed for arterial 
, 
, and pH (AVL, model 995). The P50 value of the blood gas analyser was set at 26.7 mmHg. Then a gas mixture of 12% O2 in N2 was delivered to the chamber for 20 min, and the recording chamber was opened for a final body temperature measurement. All measurements were performed between 10.00 h and 12.00 h to minimize changes in endocrine and respiratory activity associated with the circadian rhythm.
Cardiovascular measurements
Measurements of mean arterial blood pressure and heart rate were obtained in operated rats only. The extension spring and catheter were tethered to the swivel at the top of the chamber allowing free and unrestricted movement. The arterial catheter was then connected to a pressure transducer (WPI; Sarasota; FL, USA) and calibrated with a water column. The blood pressure signal was analysed on a PC by the data analysis software (Buxco Biosystem XA); heart rate was analysed on a beat-by-beat basis and mean values were logged every minute. Mean arterial blood pressure was calculated from the integrated mean of the pressure wave measured from systole to systole and logged every minute also. Cardiovascular variables were measured under normoxia and after 20 min of exposure to moderate hypoxia.
In situ hybridization for c-fos mRNA
c-fos mRNA expression was used as a functional marker of basal PVH neurone activation during normoxia (Hirooka et al. 1997; Teppema et al. 1997; Gozal et al. 1999; Berquin et al. 2000a). In a separate series of experiments, brains were harvested from 20 non-operated rats subjected to NMS (14 males, 6 females) and 30 controls (19 males, 11 females). Ventilatory activity was not recorded in these animals.
Brain tissue harvesting Rats were allowed to acclimatize to the plethysmography chamber according to the protocol described for ventilatory measurements. The chamber was then opened and the rat was deeply anaesthetized with a ketamine (Rogarsetic, 80 mg kg-1) and xylazine (Rompun, 10 mg kg-1) solution injected intraperitoneally and perfused with 0.9% saline followed by 4% paraformaldehyde (PFA) in 0.1 M sodium tetraborate buffer (PFA/borax; pH 9.5 at 4°C). Brains were removed from the skull, postfixed for 48 h in 4% PFA/borax, and then placed in 20% sucrose–4% paraformaldehyde solution for 48 h at 4°C. Frozen brains were mounted on a microtome and the hypothalamic region was cut in 30 µm coronal sections. Slices were collected in a cold cryoprotectant solution (0.05 M sodium phosphate buffer, 30% ethylene glycol and 20% glycerol) and stored at -20°C.
In situ hybridization histochemistry
Protocols for riboprobe synthesis, hybridization, and autoradiographic localization of mRNA signal were previously described (Mansi et al. 2000). The c-fos mRNA probe (2 kb) was generated from the cDNA of the rat (Dr I. Verma, Salk Institute). This fragment was subcloned into pBS-Sk+ at the EcoRI site and linearized with HindIII/T3 and SmaI/T7 for sense and antisense probes, respectively. Every fourth tissue slice was mounted onto poly-L-lysine-coated slides, desiccated under vacuum overnight, fixed in 4% paraformaldehyde for 20 min, and digested by proteinase K. Brain sections were then rinsed in sterile DEPC-water followed by a solution of 100 mM triethanolamine (pH 8.0), acetylated in 0.25% acetic anhydride in 100 mM triethanolamine, and dehydrated through graded concentrations of alcohol. After vacuum drying for a minimum of 2 h, 90 µl of hybridization mixture (107 c.p.m. ml-1) was spotted on each slide, sealed under a coverslip and incubated at 57°C overnight (
15–20 h) in a slide warmer. Coverslips were then removed and the slides were rinsed in 4 x standard saline citrate (SSC; 1 x SSC: 150 mM NaCl, 15 mM trisodium citrate buffer, pH 7.0) at room temperature. Sections were digested by RNAse A (10 mg ml-1, 37°C, 30 min), rinsed in decreasing concentrations of SSC, washed in 0.1 x SSC for 30 min at 60°C, and dehydrated through graded concentrations of alcohol. After being dried for 2 h under vacuum, the sections were exposed to X-ray film (Kodak) overnight. Radioisotope-labelled sense (control) cRNA copies were also prepared to verify the specificity of each probe. Hybridization with these probes revealed no signal in the rat brain.
Pilot studies on the potential effects of novelty stress on c-fos mRNA expression Preliminary results showed that, in male rats, PVH c-fos mRNA expression was higher in males subjected to NMS (n= 8)versus controls (n= 11). Since the recording chamber is a novel environment which could stress the animal, subgroups of male rats (NMS: n= 6; control: n= 8) were habituated to the experimental chambers 15 min per day for 5 consecutive days before brains were harvested on day 6. This adaptation procedure had no significant effect on PVH c-fos mRNA signal (P = 0.64; data not shown), and the data (adapted + nonadapted) were pooled for each group.
Corticosterone and ACTH Assays
Being aware that changes in c-fos mRNA are not necessarily accompanied by equivalent changes in protein expression, the use of PVH c-fos mRNA levels as an indicator of HPA axis activation was validated by measuring plasma ACTH and corticosterone levels. In a separate series of experiments, blood samples were collected under baseline conditions from 12 NMS rats (5 males, 7 females) and 19 controls (8 males, 11 females). Samples were collected immediately after rats were deeply anaesthetized to remove the brain. Blood was collected with a plastic syringe through the left ventricle of the heart and transferred into a EDTA vacutainer tube (Becton Dickinson). The plasma was separated by centrifugation, quickly frozen at –20°C for corticosterone and –80°C for ACTH until assayed. Corticosterone and ACTH levels were determined, respectively, by a an enzyme immunoassay (Assay Design Inc., Ann Harbour, MI, USA) and a highly specific radioimmunoassay using 125I as a tracer (Nichols Institute Diagnostics, San Clemente, CA, USA). Corticosterone detection was done with a microplate spectrophotometer (µ-Quant, Bio-Tek Instruments Inc., Winooski, VT, USA) and the 125I tracer (ACTH) was quantified with a gamma counter (Perkin Elmer Life Sciences, model 1470; Boston, MA, USA).
Data analysis
Cardio-respiratory measurements Baseline measurements of ventilatory and cardiovascular variables were obtained by averaging the last 10 min of stable recording, whereas a 5-min average was taken for each variable at the end of the hypoxic exposure. The time course of the frequency response to hypoxia was analysed in two distinct segments. The early phase was defined as the period from the onset of hypoxia (t= 0 min) to the time at which breathing frequency reached steady-state (t= 8 min). The late phase (t > 8 min) was defined as the period during which the frequency response reached steady state until the end of the hypoxic stimulus (t= 20 min; Fig. 2A and B).
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Statistical analysis
Cardio-respiratory data were analysed using repeated measure three-way ANOVA for repeated measures (hypoxic stimulus x sex x separation; Statview 5.0 SAS Institute, Cary, NC, USA). In situ hybridization and hormone data were analysed using a two-way ANOVA (sex x separation). These analyses were followed by a post hoc Fisher's test when appropriate.
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Results |
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Densitometric analysis of the c-fos mRNA signal showed that the effects of NMS are sex-specific (sex x separation: P= 0.038). In control rats, c-fos mRNA level was greater in females than in males, and neonatal maternal separation (NMS) increased c-fos mRNA expression level in the PVH of males but not females (Fig. 1A and B).
Baseline ACTH and corticosterone levels measured in control animals were comparable to those reported in other studies (Weinstock et al. 1998; Koehl et al. 1999; Dumont et al. 2000; Liu et al. 2000) indicating that ‘basal’ HPA axis activation was low. The effect of NMS on plasma ACTH levels were sex-specific also (sex x separation: P= 0.035). Plasma ACTH levels of males were higher than females (sex effect: P < 0.0001). In females, there were no differences in resting plasma levels of ACTH between control and NMS (Fig. 1C). In contrast, NMS males showed higher resting ACTH plasma level than controls (Fig. 1C).
Overall, plasma corticosterone levels measured in males were greater than in females (sex effect: P= 0.039). However, male data were highly variable and despite a trend similar to the one previously described for ACTH, there was no significant effect of NMS (Fig. 1D). Female rats showed no between-group difference in basal corticosterone levels (Fig. 1D).
Sex, neonatal maternal separation, and basal minute ventilation
Baseline (normoxic) ventilatory measurements of control animals were comparable to those reported in other studies using male Sprague–Dawley rats under similar conditions (Olson & Dempsey, 1978; Fukuda, 1991; Frappell et al. 1992; Carley et al. 1997; Peever & Stephenson, 1997). Few studies have measured ventilatory activity in female rats; between-sex comparison for similar studies is reported in Table 2. Females subjected to NMS showed a higher resting minute ventilation compared to controls (P = 0.01; Table 1) owing to a greater tidal volume (P = 0.009; Fig. 3C). These females had a lower body weight also (P = 0.0007; Table 1). These results contrast with data reported for males in which baseline minute ventilation was the same for NMS and control rats (P = 0.5; Table 1). None of the other baseline respiratory variables reported in Table 1 were affected by NMS treatment.
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Temporal analysis of the frequency response to hypoxia showed that the initial phase (0–8 min) varied according to stimulus, sex and separation (tri-factorial interaction: P= 0.022). Females subjected to NMS had a weaker hypoxic frequency response than controls (Fig. 2A). At the onset of hypoxia, NMS males showed a stronger increase of the respiratory frequency than controls (Fig. 2B).
During the late phase of the response (9–20 min), statistical analysis indicated that the effects of NMS on the frequency response to hypoxia were sex-specific (sex x separation: P= 0.003, and stimulus x separation: P= 0.03), and that the time course of the hypoxic ventilatory response varied between NMS and control (P = 0.025) in a sex-specific manner (P = 0.06). The reduced frequency response seen in the initial phase in NMS females persisted throughout the steady-state period (Fig. 2A). These data contrast with results obtained in males in which there was no between-group difference during steady state (Fig. 2B).
Sex, neonatal maternal separation, and hypoxic ventilatory response
Hypoxia had a significant effect on most variables reported in Table 1 with the exception of 
. Specifically, 
and 
decreased whereas pHa increased. After 20 min of hypoxia, body temperature decreased in all groups; however, the decrease observed in NMS females was greater than controls. Arterial pH was more alkaline in NMS males in comparison with controls (Table 1). These changes in arterial blood gases reflect the increase in ventilatory variables observed in all groups during hypoxia (stimulus effect: P < 0.0001; Fig. 3).
Both minute ventilation and breathing frequency varied according to stimulus, sex and separation (tri-factorial interaction: P= 0.06 and P= 0.006, respectively). Overall, respiratory frequency was greater in males than in females, and conversely, tidal volume was higher in females than in males (sex effect: P < 0.0001 for both; Fig. 3).
Detailed analysis of mean data obtained after 20 min of hypoxia showed that NMS had sex-specific effects on breathing frequency (sex x separation: P= 0.03 and stimulus x separation: P= 0.01; Fig. 3B and E). Minute ventilation was greater in NMS males in comparison with controls (Fig. 3D); however, no significant between-group difference was detected for frequency and tidal volume data (Figs 3E and F). In females, NMS had opposite effects on hypoxic breathing frequency (decrease; Fig. 3B) and tidal volume (increase; Fig. 2C), with no net effect on minute ventilation in comparison with controls (Fig. 3A).
To confirm these effects of NMS and sex on the hypoxic ventilatory response, selected ventilatory variables were normalized and expressed as a percentage change from baseline values. This normalization showed a significant interaction between sex and separation for minute ventilation (P = 0.007), respiratory frequency (P = 0.01), tidal volume (P = 0.05), and inspiratory flow (P = 0.02). Adult female rats subjected to NMS showed a decreased hypoxic ventilatory response relative to controls (Fig. 4A). This reduced responsiveness was mediated by a 34% decrease in the frequency response (Fig. 4A); NMS did not affect the tidal volume component of the response (Fig. 4A). These results contrast with those of NMS males in which the responsiveness to moderate hypoxia was 25% greater than controls due to the increased tidal volume response; the respiratory frequency component was unchanged by NMS (Fig. 4B). The percentage change of the ratio of tidal volume to inspiratory time (VT/TI), an index of inspiratory effort, was increased in NMS males also (Fig. 4B). This additional analysis corroborates the data previously described (absolute values) showing that the effects of NMS on the hypoxic ventilatory response were sex-specific.
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Baseline mean arterial blood pressure of control animals was comparable to those reported in other studies using male and female Sprague–Dawley rats under similar conditions (Carley et al. 1997; Bhatnagar et al. 1998; Hayward et al. 1999; Coatmellec-Taglioni et al. 2002, 2003). A three-way ANOVA indicated that mean arterial blood pressure was affected by sex and NMS (P = 0.04 and P= 0.03, respectively). Despite a suggestive trend, there was no between-group difference in resting blood pressure in females (P = 0.29; Fig. 5A). In contrast, male NMS rats were hypertensive in comparison to controls (P = 0.01; Fig. 5B). Mean arterial blood pressure decreased during hypoxia (P = 0.0007); while this response was not influenced by sex or NMS (two-factor interaction: P= 0.32 and P= 0.17, respectively).
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Discussion |
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Neonatal maternal separation and basal HPA activity
The paraventricular nucleus of the hypothalamus (PVH) is instrumental to the orchestration of the neural, behavioural, and endocrine responses to stress (Akil et al. 1999; Francis & Meaney, 1999); NMS persistently increases HPA axis responsiveness owing to impaired negative feedback sensitivity (van Oers et al. 1998; for review, see Francis et al. 1999). Neonatal maternal separation results in complex effects on behaviour and neuroendocrine function that varies significantly according to the protocols and rat strains investigated (Lehmann & Feldon, 2000). Since animal care environments can influence neurochemical profile and stress responsiveness (Beck & Luine, 2002; D'Arbe et al. 2002), the first part of our study aimed at validating the effects of performing a NMS protocol in our laboratory on resting HPA axis function. Results showing the sex-specific effects of NMS on PVH c-fos expression, ACTH and corticosterone levels are consistent with previous studies showing that alteration of HPA axis regulation by NMS is greater in males than females (Anisman et al. 1998; Francis et al. 1999; Francis & Meaney, 1999; Wigger & Neumann, 1999). While enhancement of basal HPA activity in NMS males may reflect a response to novelty stress, the fact that c-fos mRNA level remained elevated after exposing rats to five daily bouts of acclimatization to the plethysmography chamber suggests that this explanation is either unlikely or that a longer acclimatization protocol would be necessary.
Neuroendocrine mechanisms explaining why NMS had no effect in females are not fully understood but may be related to the effects of the sex steroids testosterone and oestrogens. Neonatal maternal separation also causes profound sex-related changes in central monoamine function in specific brain regions (Matthews et al. 2001). This effect, in addition to the inhibition of growth hormone secretion (Kuhn & Schanberg, 1998), is likely to contribute to the slightly lower body weight and higher resting tidal volume of NMS females since CO2 production data does not suggest that NMS affects metabolism. However, direct O2 consumption measurement would be necessary to support this interpretation further.
Neonatal maternal separation and sex-specific plasticity of the hypoxic ventilatory response
The hypoxic ventilatory response is a complex interplay between several distinct mechanisms. Among the features distinguishing these mechanisms are the stimulus characteristics (duration and intensity), the time course of the response, and the effects on the components of the ventilatory output (frequency versus tidal volume) (Powell et al. 1998). Detailed analysis of the ventilatory response during the early and late phases of the hypoxic period revealed significant sex-specific effects of NMS on the time course of the frequency response. Since the early phase (onset) of the response is commonly attributed to peripheral chemoreceptor activation (Powell et al. 1998), the present data suggest that carotid bodies of NMS males are more responsive to hypoxia than controls. Conversely, NMS would have exerted opposite (inhibitory) effects on carotid body function of NMS females in comparison with untreated rats.
During the late phase of hypoxia, the frequency response of males was not affected by NMS but was depressed in females. This part of the acute response to hypoxia is determined by overall changes in baseline ventilatory drive and central integration of relevant chemosensory afferents. Since blood gas data show no treatment-related differences in the hypoxic ventilatory drive, the reduced frequency response of NMS females suggests that, in addition to chemoreceptor function, central components of the chemoreflex loop are attenuated either at the main site of chemosensory integration (NTS) and/or other groups of premotor neurones located downstream (e.g. parabrachial nucleus, Kolliker–Fuse, ventro-lateral medulla). The specific origin and nature of these inhibitory influences on the breathing frequency response remain unknown but clearly they did not occur in NMS males. Nonetheless, the data strongly suggest that NMS affected carotid body function in a sex-specific manner.
The tidal volume component of the hypoxic ventilatory response was affected by NMS in a sex-specific fashion also. Despite showing higher values at rest, NMS females were able to increase tidal volume to the same extent as controls. The factors responsible for this change in breathing pattern are unknown, but based on blood gas data, this strategy is sufficient to maintain adequate gas exchange in the face of the reduced frequency response during hypoxic challenge in NMS females.
In males, NMS enhanced the tidal volume and concomitant inspiratory flow responses mainly account for the 25% increase in hypoxic ventilatory response in this group. This facilitation of inspiratory effort may be related to the higher level of PVN activation, since this hypothalamic nucleus sends direct neural projections on to phrenic motoneurones, and PVH activation augments diaphragmatic EMG (Yeh et al. 1997; Kc et al. 2002; Mack et al. 2002). Although whole body plethysmography is the best method currently available to measure tidal volume in awake rodents (Mortola & Frappell, 1998), its validity is the subject of much debate (Enhorning et al. 1998; Lundblad et al. 2002; Szewczak & Powell, 2003). While this limitation brings us to interpret our data cautiously, our recent results showing that male anaesthetized rats previously subjected to NMS have a greater phrenic burst amplitude response to hypoxia than controls support the tidal volume data presented here (Kinkead & Gulemetova, 2003). Together, these data indicate a clear sex-related difference in the effects of NMS on the tidal volume versus breathing frequency responses to hypoxia, thus suggesting that the sensitivity to steroid hormone fluctuations caused by our experimental protocol varies substantially amongst the different components of the respiratory control system.
Neonatal maternal separation and development of blood pressure regulation
Mean arterial blood pressure of adult NMS males is roughly 20% higher than controls; heart rate was not affected by this treatment. Sex differences in blood pressure are well documented in hypertensive rat models: in the spontaneously hypertensive rat strain (SHR), males have a higher blood pressure than females at a similar age (Reckelhoff et al. 2000). Based on the wealth of literature on the potential link between changes in hypothalamic function and hypertension (De Wardener, 2001), it is tempting to propose that NMS-related enhancement of HPA function is at the basis of the hypertension observed in NMS males in this study. Increase in PVH function leads to an increase in sympathetic outflow which, in turn, will result in hypertension (De Wardener, 2001). Since carotid body function modulates sympathetic tone, the ventilatory data suggesting enhancement of carotid body function in NMS males would suggest that sympathetic outflow is greater in NMS males than controls. While this interplay between chemoreflexes and blood pressure regulation was not addressed directly in our study, the fact that carotid body and ventilatory responses to hypoxia are greater in spontaneously hypertensive rats than in normotensive controls is consistent with this interpretation (Fukuda et al. 1987; Hayward et al. 1999).
Perspective
Neonatal maternal separation has extensive effects on central nervous system development as the tactile, olfactory, and auditory stimuli provided by the mother during mother–pup interactions are essential to normal growth and development (Kuhn & Schanberg, 1998). While the long-term consequences of depriving pups from maternal stimuli have been associated mainly with psychological development (Kuhn & Schanberg, 1998), our results show that the long-lasting effects of NMS on CNS development also include vital homeostatic functions such as cardio-respiratory regulation. Based on the fact that glucocorticoid overexposure during early life leads to hypertension in rat (O'Regan et al. 2001), the role of these important modulators of gene expression must be considered in the programming of the hypoxic ventilatory response also.
While the results of the present study may be viewed as descriptive, they raise several important questions concerning the proximal causes and mechanisms of these intriguing manifestations of cardio-respiratory plasticity. Since sex-specific (male) hypertension and increased peripheral chemoreceptor function are key features of human cardio-respiratory disorders such as sleep-related breathing disorders (Young et al. 1993; Khoo, 1999; Kara et al. 2003), NMS may be a valuable model to investigate the mechanisms underlying this pathology.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Anisman H, Zaharia MD, Meaney MJ & Merali Z (1998). Do early-life events permanently alter behavioral and hormonal responses to stressors?Int J Dev Neurosci 16, 149–164.[CrossRef][Medline]
Beck KD & Luine VN (2002). Sex differences in behavioral and neurochemical profiles after chronic stress. Role of housing conditions. Physiol Behav 75, 661–673.[CrossRef][Medline]
Berquin P, Bodineau L, Gros F & Larnicol N (2000a). Brainstem and hypothalamic areas involved in respiratory chemoreflexes: a Fos study in adult rats. Brain Res 857, 30–40.[CrossRef][Medline]
Berquin P, Cayetanot F, Gros F & Larnicol N (2000b). Postnatal changes in Fos-like immunoreactivity evoked by hypoxia in the rat brainstem and hypothalamus. Brain Res 877, 149–159.[CrossRef][Medline]
Bhatnagar S, Dallman MF, Roderick RE, Basbaum AI & Taylor BK (1998). The effects of prior chronic stress on cardiovascular responses to acute restraint and formalin injection. Brain Res 797, 313–320.[CrossRef][Medline]
Carley DW, Trbovic SM, Bozanich A & Radulovacki M (1997). Cardiopulmonary control in sleeping Sprague-Dawley rats treated with hydralazine. J Appl Physiol 83, 1954–1961.
Carroll JL (2003). Plasticity in respiratory motor control: Invited review: developmental plasticity in respiratory control. J Appl Physiol 94, 375–389.
Coatmellec-Taglioni G, Dausse JP, Giudicelli Y & Ribiere C (2002). Gender difference in diet-induced obesity hypertension: implication of renal alpha2-adrenergic receptors. Am J Hypertens 15, 143–149.[CrossRef][Medline]
Coatmellec-Taglioni G, Dausse JP, Giudicelli Y & Ribiere C (2003). Sexual dimorphism in cafeteria diet-induced hypertension is associated with gender-related difference in renal leptin receptor down-regulation. J Pharmacol Exp Ther 305, 362–367.
D'Arbe M, Einstein R & Lavidis NA (2002). Stressful animal housing conditions and their potential effect on sympathetic neurotransmission in mice. Am J Physiol Regul Integr Comp Physiol 282, R1422–R1428.
De Wardener HE (2001). The hypothalamus and hypertension. Physiol Rev 81, 1599–1658.
Dumont EC, Kinkead R, Trottier J, Gosselin I & Drolet G (2000). Effect of chronic psychogenic stress exposure on enkephalin neuronal activity and expression in the rat hypothalamic paraventricular nucleus. J Neurochem 75, 2200–2211.[CrossRef][Medline]
Enhorning G, van Schaik S, Lundgren C & Vargas I (1998). Whole-body plethysmography, does it measure tidal volume of small animals?Can J Physiol Pharmacol 76, 945–951.[CrossRef][Medline]
Erickson JT, Mayer C, Jawa A, Ling L, Olson EB Jr, Vidruk EH, Mitchell GS & Katz DM (1998). Chemoafferent degeneration and carotid body hypoplasia following chronic hyperoxia in newborn rats. J Physiol 509, 519–526.
Francis DD, Caldji C, Champagne F, Plotsky PM & Meaney MJ (1999). The role of corticotropin-releasing factor – norepinephrine systems in mediating the effects of early experience on the development of behavioral and endocrine responses to stress. Biol Psychiatry 46, 1153–1166.[CrossRef][Medline]
Francis DD & Meaney MJ (1999). Maternal care and the development of stress responses. Curr Opin Neurobiol 9, 128–134.[CrossRef][Medline]
Frappell P, Lanthier C, Baudinette RV & Mortola JP (1992). Metabolism and ventilation in acute hypoxia: a comparative analysis in small mammalian species. Am J Physiol 262, R1040–R1046.[Medline]
Fukuda Y (1991). Maintenance of ventilatory control by CO2 in the rat during growth and aging. Pflugers Arch 419, 38–42.[CrossRef][Medline]
Fukuda Y, Sato A & Trezebski A (1987). Carotid chemoreceptor discharge responses to hypoxia and hypercapnia in normotensive and spontaneously hypertensive rats. J Auton Nerv Syst 19, 1–11.[CrossRef][Medline]
Gozal D, Xue YD & Simakajornboon N (1999). Hypoxia induces c-Fos protein expression in NMDA but not AMPA glutamate receptor labeled neurons within the nucleus tractus solitarii of the conscious rat. Neurosci Lett 262, 93–96.[CrossRef][Medline]
Hayward LF, Johnson AK & Felder RB (1999). Arterial chemoreflex in conscious normotensive and hypertensive adult rats. Am J Physiol 276, H1215–H1222.[Medline]
Herman JP & Cullinan WE (1997). Neurocircuitry of stress: central control of the hypothalamo-pituitary-adrenocortical axis. Trends Neurosci 20, 78–84.[CrossRef][Medline]
Hirooka Y, Polson JW, Potts PD & Dampney RA (1997). Hypoxia-induced Fos expression in neurons projecting to the pressor region in the rostral ventrolateral medulla. Neuroscience 80, 1209–1224.[CrossRef][Medline]
Joseph V, Soliz J, Pequignot J, Sempore B, Cottet-Emard JM, Dalmaz Y, Favier R, Spielvogel H & Pequignot JM (2000). Gender differentiation of the chemoreflex during growth at high altitude: functional and neurochemical studies. Am J Physiol Regul Integr Comp Physiol 278, R806–R816.
Kara T, Narkiewicz K & Somers VK (2003). Chemoreflexes – physiology and clinical implications. Acta Physiol Scand 177, 377–384.[CrossRef][Medline]
Kc P, Haxhiu MA, Trouth CO, Balan KV, Anderson WA & Mack SO (2002). CO2-induced c-Fos expression in hypothalamic vasopressin containing neurons. Respir Physiol 129, 289–296.[CrossRef][Medline]
Khoo MCK (1999). Periodic breathing and central apnea. In Lung Biology in Health and Disease, vol. 135, ed. Altose MD & Kawakami, pp. 203–250. Marcel Dekker, New York, Basel.
Kinkead R, Dupenloup L & Gulemetova R (2001). Stress-induced attenuation of the hypercapnic ventilatory response in awake rats. J Appl Physiol 90, 1729–1735.
Kinkead R & Gulemetova R (2003). Neonatal maternal separation enhances time-dependent phrenic responses to hypoxia in rat. FASEB J 17, A1297.
Koehl M, Darnaudery M, Dulluc J, Van Reeth O, Le Moal M & Maccari S (1999). Prenatal stress alters circadian activity of hypothalamo-pituitary-adrenal axis and hippocampal corticosteroid receptors in adult rats of both gender. J Neurobiol 40, 302–315.[CrossRef][Medline]
Kuhn CM & Schanberg SM (1998). Responses to maternal separation: mechanisms and mediators. Int J Dev Neurosci 16, 261–270.[Medline]
Ladd CO, Huot RL, Thrivikraman KV, Nemeroff CB, Meaney MJ & Plotsky PM (2000). Long-term behavioral and neuroendocrine adaptations to adverse early experience. Prog Brain Res 122, 81–103.[Medline]
Lehmann J & Feldon J (2000). Long-term biobehavioral effects of maternal separation in the rat: consistent or confusing?Rev Neurosci 11, 383–408.[Medline]
Ling L, Olson EB Jr, Vidruk EH & Mitchell GS (1996). Attenuation of the hypoxic ventilatory response in adult rats following one month of perinatal hyperoxia. J Physiol 495, 561–571.
Ling L, Olson EB Jr, Vidruk EH & Mitchell GS (1997). Developmental plasticity of the hypoxic ventilatory response. Respir Physiol 110, 261–268.[CrossRef][Medline]
Liu D, Caldji C, Sharma S, Plotsky PM & Meaney MJ (2000). Influence of neonatal rearing conditions on stress-induced adrenocorticotropin responses and norepinepherine release in the hypothalamic paraventricular nucleus. J Neuroendocrinol 12, 5–12.[CrossRef][Medline]
Lundblad LK, Irvin CG, Adler A & Bates JH (2002). A reevaluation of the validity of unrestrained plethysmography in mice. J Appl Physiol 93, 1198–1207.
Mack SO, Kc P, Wu M, Coleman BR, Tolentino-Silva FP & Haxhiu MA (2002). Paraventricular oxytocin neurons are involved in neural modulation of breathing. J Appl Physiol 92, 826–834.
Mansi JA, Laforest S & Drolet G (2000). Effect of stress exposure on the activation pattern of enkephalin-containing perikarya in the rat ventral medulla. J Neurochem 74, 2568–2575.[CrossRef][Medline]
Matthews K, Dalley JW, Matthews C, Tsai TH & Robbins TW (2001). Periodic maternal separation of neonatal rats produces region- and gender-specific effects on biogenic amine content in postmortem adult brain. Synapse 40, 1–10.[CrossRef][Medline]
Mortola JP & Dotta A (1992). Effects of hypoxia and ambient temperature on gaseous metabolism of newborn rats. Am J Physiol 263, R267–R272.[Medline]
Mortola JP & Frappell PB (1998). On the barometric method for measurements of ventilation, and its use in small animals. Can J Physiol Pharmacol 76, 937–944.[CrossRef][Medline]
Mortola JP & Saiki C (1996). Ventilatory response to hypoxia in rats: gender differences. Respir Physiol 106, 21–34.[CrossRef][Medline]
O'Regan D, Welberg LL, Holmes MC & Seckl JR (2001). Glucocorticoid programming of pituitary-adrenal function: mechanisms and physiological consequences. Semin Neonatol 6, 319–329.[CrossRef][Medline]
van Oers HJ, de Kloet ER & Levine S (1998). Early vs. late maternal deprivation differentially alters the endocrine and hypothalamic responses to stress. Brain Res Dev Brain Res 111, 245–252.[Medline]
Okubo S & Mortola JP (1988). Long-term respiratory effects of neonatal hypoxia in the rat. J Appl Physiol 64, 952–958.
Okubo S & Mortola JP (1990). Control of ventilation in adult rats hypoxic in the neonatal period. Am J Physiol 259, R836–R841.[Medline]
Olson EB Jr & Dempsey JA (1978). Rat as a model for humanlike ventilatory adaptation to chronic hypoxia. J Appl Physiol 44, 763–769.
Pan LG, Forster HV, Martino P, Strecker PJ, Beales J, Serra A, Lowry TF, Forster MM & Forster AL (1998). Important role of carotid afferents in control of breathing. J Appl Physiol 85, 1299–1306.
Peever JH & Stephenson R (1997). Day-night differences in the respiratory response to hypercapnia in awake adult rats. Respir Physiol 109, 241–248.[CrossRef][Medline]
Powell FL, Milsom WK & Mitchell GS (1998). Time domains of the hypoxic ventilatory response. Respir Physiol 112, 123–134.[CrossRef][Medline]
Reckelhoff JF, Zhang H & Srivastava K (2000). Gender differences in development of hypertension in spontaneously hypertensive rats: Role of the renin-angiotensin system. Hypertension 35, 480–483.
Rezzonico R & Mortola JP (1989). Respiratory adaptation to chronic hypercapnia in newborn rats. J Appl Physiol 67, 311–315.
Sanchez MM, Ladd CO & Plotsky PM (2001). Early adverse experience as a developmental risk factor for later psychopathology: evidence from rodent and primate models. Dev Psychopathol 13, 419–449.[CrossRef][Medline]
Schlenker E, Barnes L, Hansen S & Martin D (2001). Cardiorespiratory and metabolic responses to injection of bicuculline into the hypothalamic paraventricular nucleus (PVN) of conscious rats. Brain Res 895, 33–40.[CrossRef][Medline]
Schlenker EH & Goldman M (1986). Aspartic acid administered neonatally affects ventilation of male and female rats differently. J Appl Physiol 61, 780–784.
Shair HN & Myers MM (1997). Effects of combined carotid sinus and aortic depressor nerve denervations in neonatal rat pups. Biol Neonate 71, 251–264.[Medline]
Swanson LW (1992). Brain maps: structure of the rat brain. Elsevier, Amsterdam.
Szewczak JM & Powell FL (2003). Open-flow plethysmography with pressure-decay compensation. Respir Physiolo Neurobiol 134, 57–67.[CrossRef][Medline]
Teppema LJ, Veening JG, Kranenburg A, Dahan A, Berkenbosch A & Olievier C (1997). Expression of c-fos in the rat brainstem after exposure to hypoxia and to normoxic and hyperoxic hypercapnia. J Comp Neurol 388, 169–190.[CrossRef][Medline]
Weinstock M, Poltyrev T, Schorer-Apelbaum D, Men D & McCarty R (1998). Effect of prenatal stress on plasma corticosterone and catecholamines in response to footshock in rats. Physiol Behav 64, 439–444.[CrossRef][Medline]
Wigger A & Neumann ID (1999). Periodic maternal deprivation induces gender-dependent alterations in behavioral and neuroendocrine responses to emotional stress in adult rats. Physiol Behav 66, 293–302.[CrossRef][Medline]
Yeh ER, Erokwu B, LaManna JC & Haxhiu MA (1997). The paraventricular nucleus of the hypothalamus influences respiratory timing and activity in the rat. Neurosci Lett 232, 63–66.[CrossRef][Medline]
Young T, Palta M, Dempsey J, Skatrud J, Weber S & Badr S (1993). The occurrence of sleep-disordered breathing among middle-aged adults. N Engl J Med 328, 1230–1235.
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Statistically different from corresponding control value (P < 0.05). Note that all values are different from baseline (P < 0.05); however, no symbols are shown for simplicity.
. Data are compared between controls (filled circles; males: n= 25 and females: n= 15) and rats previously subjected to NMS (open triangles; males: n= 15 and females: n= 18). Values are expressed as means ±S.E.M.
and expressed as a percentage changes from normoxic baseline values. Filled bars represent control animals (males: n= 25 and females: n= 15) and open bars represent rats subjected to NMS (males: n= 15 and females: n= 18). Data are expressed as means ±S.E.M.
statistically different from corresponding control value (P = 0.06).
