Conscious Adult Rats Research Articles

Brainstem astrocytes regulate breathing and may affect arousal state in rats

Variations in arousal levels can impact respiratory patterns. The mechanisms by which breathing behaviors can influence arousal state is not fully understood. In this study, we investigated the role of astrocytes in the preBötzinger complex (preBötC) in modulating arousal states via breathing in adult conscious rats. Using viral vector tools, we selectively interfered with astrocytic signaling in the preBötC. Rats with inhibited astrocytic signaling exhibited slower breathing rates and behaviors indicative of a calmer state, whereas enhanced purinergic signaling in preBötC astrocytes led to faster breathing and heightened arousal. Our findings reveal a key role for an astrocyte-mediated mechanism in the preBötC that influences both respiratory behaviors and higher-order brain functions like arousal, suggesting a bidirectional link between breathing behaviors and mental states.

Effect of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, on behavioral and physiological parameters.

The role of brain derived nitric oxide in the physiology and behavior remains disputable. One of the reasons of the controversies might be systemic side effects of nitric oxide synthase inhibitors. Therefore, under nNOS inhibition by 7-nitroindazole (7-NI) we carried out recordings of blood gasses, blood pressure and spontaneous EEG in conscious adult rats. Locomotion and spontaneous behavior were assessed in an open field. In addition skilled walking and limb coordination were evaluated using a ladder rung walking test. The blood gas analysis revealed a significant increase in pCO(2) 180 min and 240 min after the application of 7-NI. The power and entropy decreased simultaneously with a shift of the mean frequency of the spontaneous EEG toward slow oscillations after 7-NI treatment. The thresholds of evoked potentials underwent a significant drop and a trend towards a slight increase in the I-O curve slope was observed. 7-NI significantly suppressed open field behavior expressed as distance moved, exploratory rearing and grooming. As for the ladder rung walking test the 7-NI treated animals had more errors in foot placement indicating impairment in limb coordination. Therefore our findings suggest that 7-NI increased cortical excitability and altered some physiological and behavioral parameters.

Central hydrogen sulphide mediates ventilatory responses to hypercapnia in adult conscious rats.

Hydrogen sulphide (H2S) is endogenously produced and plays an important role as a modulator of neuronal functions; however, its modulatory role in the central CO2 chemoreception is unknown. The aim of the present study was to assess the role of endogenously produced H2S in the ventilatory response to hypercapnia in adult conscious rats. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) inhibitors (aminooxyacetate: AOA and propargylglycine: PAG respectively) and a H2S donor (sodium sulphide: Na2S) were microinjected into the fourth ventricle (4V). Ventilation (V̇(E)), oxygen consumption (V̇O2) and body temperature were recorded before (room air) and during a 30-min CO2 exposure (hypercapnia, 7% CO2). Endogenous H2S levels were measured in the nucleus tractus solitarius (NTS). Microinjection of Na2S (H2S donor), AOA (CBS inhibitor) or PAG (CSE inhibitor) did not affect baseline of the measured variables compared to control group (vehicle). In all experimental groups, hypercapnia elicited an increase in V̇(E). However, AOA microinjection, but not PAG, attenuated the ventilatory response to hypercapnia (P < 0.05), whereas Na2S elicited a slight, not significant, enhancement. Moreover, endogenous H2S levels were found higher in the NTS after hypercapnia (P < 0.05) compared to room air (normoxia) condition. There are a few reports on the role of gaseous transmitters in the control of breathing. Importantly, the present data suggest that endogenous H2S via the CBS-H2S pathway mediates the ventilatory response to hypercapnia playing an excitatory role.

Effects of acute intermittent hypoxia on sympathetic and expiratory activities in rats (872.8)

The exposure to acute intermittent hypoxia (AIH) produces a long‐lasting increase of phrenic and sympathetic activities in anesthetized rats. In the present study we aimed to evaluate the effects of AIH on baseline expiratory motor activity as well as the role of retrotrapezoid nucleus in the AIH‐induced sympathetic and respiratory changes. In the in situ working heart‐brainstem preparations of juvenile rats (n=4), AIH exposure (10 x 5‐7% O2 for 30 s, every 5 min) increased baseline phrenic (Δ: 20±6%), abdominal (Δ: 65±30%) and sympathetic (Δ: 89±48%) nerve activities (P&lt;0.05) that was maintained for at least 60 min after the last hypoxic episode. In conscious adult rats (n=9), AIH evoked an increase in baseline arterial pressure (Δ: 7±2 mmHg), associated with enhanced sympathetic‐ and respiratory‐related variabilities (P&lt;0.05). In adult urethane‐anesthetized rats with intact vagus (n=5), AIH increased baseline arterial pressure (Δ: 9±1 mmHg) and abdominal muscle contraction amplitude (Δ: 412±117%), which were reverted by pharmacological inhibition of retrotrapezoid nucleus (P&lt;0.05). These data indicate that AIH promotes a sustained increase of expiratory activity, in addition to inspiratory and sympathetic activities. Moreover, AIH‐induced sympathetic and expiratory long‐term facilitation may involve, at least in part, the activation of the retrotrapezoid nucleus.Grant Funding Source: Supported by FAPESP (09/54888‐7; 11/20040‐1) and CNPq

Development of attenuated baroreflexes in obese Zucker rats coincides with impaired activation of nucleus tractus solitarius

Adult obese Zucker rats (OZR; >12 wk) develop elevated sympathetic nerve activity (SNA) and mean arterial pressure (MAP) with impaired baroreflexes compared with adult lean Zucker rats (LZR) and juvenile OZR (6-7 wk). In adult OZR, baroreceptor afferent nerves respond normally to changes in MAP, whereas electrical stimulation of baroreceptor afferent fibers produces smaller reductions in SNA and MAP compared with LZR. We hypothesized that impaired baroreflexes in OZR are linked to reduced activation of brain stem sites that mediate baroreflexes. In conscious adult rats, a hydralazine (HDZ)-induced reduction in MAP evoked tachycardia that was initially blunted in OZR, but equivalent to LZR within 5 min. In agreement, HDZ-induced expression of c-Fos in the rostral ventrolateral medulla (RVLM) was comparable between groups. In contrast, phenylephrine (PE)-induced rise in MAP evoked markedly attenuated bradycardia with dramatically reduced c-Fos expression in the nucleus tractus solitarius (NTS) of adult OZR compared with LZR. However, in juvenile rats, PE-induced hypertension evoked comparable bradycardia in OZR and LZR with similar or augmented c-Fos expression in NTS of the OZR. In urethane-anesthetized rats, microinjections of glutamate into NTS evoked equivalent decreases in SNA, heart rate (HR), and MAP in juvenile OZR and LZR, but attenuated decreases in SNA and MAP in adult OZR. In contrast, microinjections of glutamate into the caudal ventrolateral medulla, a target of barosensitive NTS neurons, evoked comparable decreases in SNA, HR, and MAP in adult OZR and LZR. These data suggest that OZR develop impaired glutamatergic activation of the NTS, which likely contributes to attenuated baroreflexes in adult OZR.

Microarray analysis of gene expression after electrical stimulation of the dura mater surrounding the superior sagittal sinus in conscious adult rats

Background The molecular and cellular origins of migraine headache are among the most complex problems in contemporary neurology. Up to now the pathogenesis of migraine still remains unclearly defined. The objective of this study was to explore new factors that may be related to the mechanism of migraine. Methods The present study performed a comprehensive analysis of gene expression in the trigeminal nucleus caudalis induced by electrical stimulation of dura mater surrounding the superior sagittal sinus in conscious rats using microarray analysis followed by quantitative real-time reverse-transcribed polymerase chain reaction (qRT-PCR) verification. Student’s two sample t-test was employed when two groups were compared. A P value &lt;0.05 was considered to be statistically significant. Results Comparing the placebo and the electrical stimulation groups, 40 genes were determined to be significantly differentially expressed. These significantly differentially expressed genes were involved in many pathways, including transporter activity, tryptophan metabolism, G protein signaling, kinase activity, actin binding, signal transducer activity, anion transport, protein folding, enzyme inhibitor activity, coenzyme metabolism, binding, ion transport, cell adhesion, metal ion transport, oxidoreductase activity, mitochondrion function, and others. Most of the genes were involved in more than 2 pathways. Of particular interest is the up-regulation of Phactr3 and Akap5 and the down-regulation of Kdr. Conclusion These findings may provide important clues for a better understanding of the molecular mechanism of migraine.

Phox2b‐expressing retrotrapezoid neurons and the integration of central and peripheral chemosensory control of breathing in conscious rats

Chemoreception is the classic mechanism by which the brain regulates breathing in response to changes in tissue CO2/H(+). A brainstem region called the retrotrapezoid nucleus (RTN) contains a population of Phox2b-expressing glutamatergic neurons that appear to function as important chemoreceptors. In the present study, we ask whether the destruction of a type of pH-sensitive interneuron that expresses the transcription factor Phox2b and is non-catecholaminergic (Phox2b(+)TH(-)) could affect breathing in conscious adult rats. The injection of substanceP (1nmol in a volume of 50nl) into the RTN increased respiratory frequency, tidal volume, minute ventilation and mean arterial pressure. Bilateral injections of the toxin substanceP conjugated with saporin (SSP-SAP) into the RTN destroyed Phox2b(+)TH(-) neurons but spared facial motoneurons, catecholaminergic and serotonergic neurons and the ventral respiratory column caudal to the facial motor nucleus. Bilateral inhibition of RTN neurons with SSP-SAP (0.6ng in 30nl) reduced resting ventilation and the increase in ventilation produced by hypercapnia (7% CO2) in conscious rats with or without peripheral chemoreceptors. In anaesthetized rats with bilateral lesions of around 90% of the Phox2b(+)TH(-) neurons, acute activation of the Bötzinger complex, the pre-Bötzinger complex or the rostral ventral respiratory group with NMDA (5pmol in 50nl) elicited normal cardiorespiratory output. In conclusion, the destruction of the Phox2b(+)TH(-) neurons is a plausible cause of the respiratory deficits observed after injection of SSP-SAP into the RTN. Our results also suggest that RTN neurons activate facilitatory mechanisms important to the control of breathing in resting or hypercapnic conditions in conscious adult rats.

Serotonergic neurons in the nucleus raphé obscurus are not involved in the ventilatory and thermoregulatory responses to hypoxia in adult rats

The medullary raphé is an important component of the central respiratory network, playing a key role in CO2 central chemoreception. However, its participation in hypoxic ventilatory responses is less understood. In the present study, we assessed the role of nucleus raphé obscurus (ROb), and specifically 5-HT neurons confined in the ROb, on ventilatory and thermoregulatory responses to hypoxia. Chemical lesions of the ROb were performed using either ibotenic acid (non-specific lesion; control animals received PBS) or anti-SERT-SAP (5-HT specific lesion; control animals received IgG-SAP). Ventilation (V˙E; whole body plethysmograph) and body temperature (Tb; data loggers) were measured during normoxia (21% O2, N2 balance) and hypoxia exposure (7% O2, N2 balance, 1h) in conscious adult rats. Ibotenic acid or anti-SERT-SAP-induced lesions did not affect baseline values of V˙E and Tb. Similarly, both lesion procedures did not alter the ventilatory or thermoregulatory responses to hypoxia. Although evidence in the literature suggests a role of the rostral medullary raphé in hypoxic ventilatory responses, under the present experimental conditions our data indicate that caudal medullary raphé (ROb) and its 5-HT neurons neither participate in the tonic maintenance of breathing nor in the ventilatory and thermal responses to hypoxia.

Pressor response to l-cysteine injected into the cisterna magna of conscious rats involves recruitment of hypothalamic vasopressinergic neurons

The sulfur-containing non-essential amino acid L-cysteine injected into the cisterna magna of adult conscious rats produces an increase in blood pressure. The present study examined if the pressor response to L-cysteine is stereospecific and involves recruitment of hypothalamic vasopressinergic neurons and medullary noradrenergic A1 neurons. Intracisternally injected D-cysteine produced no cardiovascular changes, while L-cysteine produced hypertension and tachycardia in freely moving rats, indicating the stereospecific hemodynamic actions of L-cysteine via the brain. The double labeling immunohistochemistry combined with c-Fos detection as a marker of neuronal activation revealed significantly higher numbers of c-Fos-positive vasopressinergic neurons both in the supraoptic and paraventricular nuclei and tyrosine hydroxylase containing medullary A1 neurons, of L-cysteine-injected rats than those injected with D-cysteine as iso-osmotic control. The results indicate that the cardiovascular responses to intracisternal injection of L-cysteine in the conscious rat are stereospecific and include recruitment of hypothalamic vasopressinergic neurons both in the supraoptic and paraventricular nuclei, as well as of medullary A1 neurons. The findings may suggest a potential function of L-cysteine as an extracellular signal such as neuromodulators in central regulation of blood pressure.

Gap junction blockade in the locus coeruleus (LC) decreases the hypercapnic ventilatory response

The LC has an important role in central chemoreception and its contribution decreases with development. In neurons from young rats (P9 or younger), the % of LC neurons that increase firing rate in response to hypercapnia is 85% while it is only about 45% in neurons from rats P10 or older. Carbenoxolone (CARB ‐ gap junction blocker) has very little effect on this response in neurons from the youngest rats, with the % responding dropping only to 70% in the presence of CARB. However, CARB decreases the % responding by half in older animals, dropping it to 21%. We evaluated the participation of gap junctions in the CO2 ventilatory response in conscious adult rats by bilaterally microinjecting CARB (1mM or 3mM / 0.1 μL) or vehicle (artificial cerebrospinal fluid) into the LC of Wistar rats. Bilateral gap junction blockade in LC neurons did not affect resting ventilation. However, the increase in ventilation produced by hypercapnia (7% CO2) was reduced by 20% after CARB injection (1904.5 ± 95.2 in vehicle group vs 1518.0 ± 76.1 mL/kg/min in CARB 3mM group and 1468.3 ± 122.2 mL/kg/min in CARB 1mM) due largely to a decrease in respiratory frequency. The results suggest that gap junctions modulate the hypercapnic ventilatory response, but that they are not involved in respiratory control during normocapnia, in the LC of rats older than P10.Financial support: INCT‐Fisiologia Comparada, FAPESP, CNPq, and NIH Grant HL56683.

Nociceptive behaviors were induced by electrical stimulation of the dura mater surrounding the superior sagittal sinus in conscious adult rats and reduced by morphine and rizatriptan benzoate

The trigeminovascular nociception induced by electrical stimulation of the dura mater surrounding the superior sagittal sinus in anesthetized animals has been widely used as a model for investigation of the pathophysiology of vascular headache such as migraine. However, little is known whether pain behaviors can be induced using this model in conscious animals. Thus, to establish a new model of trigeminovascular nociception in conscious animals and to examine the effects of morphine and rizatriptan benzoate on nociceptive behaviors in this new model, we electrically stimulated the dura mater surrounding the superior sagittal sinus. We found that grooming and head-flick activities were altered partially in a frequency-dependent way and that frequencies ranging from 10 to 20 Hz more easily provoked these behaviors. Moreover, we also demonstrated that these behaviors were reduced by morphine and rizatriptan benzoate. Thus, this new model will provide a useful and appropriate tool to directly assess changes in the intensity of pain for further investigation of pathophysiological mechanisms of migraine in conscious animals.

Perinatal taurine exposure alters renal potassium excretion in adult conscious rats

Perinatal taurine exposure alters renal hemodynamics and sodium excretion in adult rats. This study further tests the effect of perinatal taurine on renal potassium excretion in adult conscious rats. Female Sprague‐Dawley rats were fed normal rat chow and given water alone (C), 3% beta‐alanine in water (taurine depletion, TD) or 3% taurine in water (taurine supplementation, TS), either from conception until delivery (fetal period; TDF or TSF) or from delivery until weaning (lactation period; TDL or TSL). In Experiment 1, after weaning, male offspring were fed normal rat chow and tap water. In Experiment 2, female pups were treated similarly, but from conception until weaning. At 7–8 weeks of age, renal potassium excretion at rest and after an acute saline load (5% of body weight) was studied in conscious, restrained rats. All male groups displayed similar renal potassium excretion. TSF increased fractional potassium excretion at rest but not in response to saline load, whereas TDF did the opposite. Plasma potassium levels were lower only at rest in TSF but at rest and during the challenge in TDF. Their glomerular filtration rates did not significantly different among groups. In female offspring, perinatal taurine supplementation decreased renal potassium excretion, glomerular filtration rate, and plasma potassium concentration at rest and after the saline load. It is unlikely that increased tubular reabsorption caused these effects, since the fractional potassium excretion significantly increased, especially at rest. Perinatal taurine depletion had no significant effect on renal potassium excretion in the female offspring. The present data suggest that perinatal taurine exposure influences renal potassium excretion in the adult rats.

Role of nitric oxide in regulating the activation of aldose reductase in the ischemic heart

Aldose reductase (AR) catalyzes the reduction of reactive aldehydes derived from lipid peroxidation. Our previous studies show that in the heart, AR is activated by ischemia; however the underlying mechanisms remain unclear. We tested the hypothesis that ischemic activation of AR is mediated by NO. For this, adult conscious rats were subjected to coronary occlusion in situ. Treatment with the iNOS inhibitor, l-NAME (50 mg/kg, i.p.) abolished ischemia-induced AR activation as well as sorbitol accumulation in both, ischemic and reperfused hearts.

Chronic inhibition of phosphoinositide‐3‐kinase (PI3K) in the nucleus of the solitary tract (NTS) of hypertensive rats increases blood pressure

The NTS is the site of termination of baroreceptor primary afferents and a major regulator of arterial pressure. Our previous work indicated an increase in PI3K signalling in brainstem/hypothalamic neuronal cultures from spontaneously hypertensive (SHRs) compared to normotensive Wistar Kyoto rats (WKYs; Yang & Raizada, 1999; Veerasingham et al. 2005). To investigate the functional implications of this, we chronically blocked PI3K activity in the NTS by overexpressing a dominant negative of the p85α regulatory subunit of PI3K (DNp85α) while measuring changes in mean arterial pressure (MAP) in both SHR and WKY conscious adult rats using radio telemetry. Transgene expression was confirmed immunohistochemically post hoc. In SHR, but not WKY, MAP increased from 146±3 to 169±9 mmHg three weeks post gene delivery (n=6; P<0.05) persisting over five weeks, with no change in the spontaneous baroreflex gain (sBRG) or heart rate (HR). There was no change in MAP, sBRG and HR in either WKYs or in the control animals expressing DNp85α and eGFP respectively. We conclude that in SHR, but not WKY, the endogenous PI3K signalling acts to regulate arterial pressure. NIH grant #HL33610.

Ontogeny of respiratory sensitivity and tolerance to the mu-opioid agonist fentanyl in rat

Whereas developmental changes in analgesic sensitivity and tolerance to the mu-opioid agonist fentanyl have been reported, knowledge of respiratory responses to that drug is lacking. Using 7- and 14-day-old (P7, P14) and adult conscious rats, we first established, using whole body plethysmography, the fentanyl dose that decreased minute ventilation by 50% (ED50) at each age. ED50 increased with postnatal age (40, 60 and 120 μg/kg sc, respectively), indicating a high sensitivity to fentanyl in the youngest rats that decreased with maturation. In separate rat groups of the 3 ages, we injected each ED50 dose, once a day, for several consecutive days, until tolerance was established. Tolerance was defined as a reduction in respiratory depression from 50% to 75% of baseline. All age groups reached tolerance in minute ventilation, respiratory frequency, tidal volume and instantaneous flow (equivalent to respiratory drive). The P14 rat pups attained tolerance more rapidly (at 2.6 days) than did either the younger (5.1 days) or the adult rats (4.4 days). These results indicate that respiratory sensitivity and tolerance to fentanyl in rat vary in a distinct manner during maturation.

Blunted central bromocriptine-induced tachycardia in conscious, malnourished rats.

Bromocriptine-induced tachycardia, persisting after adrenalectomy, is mediated by central dopamine D2 receptor stimulation through activation of the sympathetic outflow to the heart. The present study investigated the effects of malnutrition during pregnancy on bromocriptine-induced tachycardia in adult conscious rats. Malnourished rats were obtained by feeding dams a multideficient diet (providing 8% protein) during mating and pregnancy. Birth weight was significantly reduced in malnourished rats when compared to control rats born to dams fed standard commercially diet (23% protein) during mating and pregnancy. Baseline mean aortic pressure and heart rate in malnourished rats were comparable to those of well-nourished rats. Tachycardia (33+/-9 beats/min.), but not the hypotensive response to intravenous bromocriptine (150 microg/kg) was significantly reduced in malnourished rats, compared with control rats (70+/-10 beats/min.). In malnourished rats, pretreatment with intravenous domperidone (500 microg/kg) blocked the bromocriptine-induced hypotension, without affecting the tachycardia. Neither cardiac vagal (40+/-6 beats/min.) nor sympathetic tone (76+/-6 beats/min.) was significantly altered by multideficient diet-induced malnutrition (51+/-6 and 67+/-10 beats/min., respectively). In isolated perfused heart preparations from malnourished rats, positive inotropic response to isoproterenol (10-8 to 10-4 M) was not significantly different compared to that in control rats. In summary, malnutrition during foetal life blunted the bromocriptine-induced tachycardia, an effect that could be related to central dopamine D2 receptor desensitization rather than to impairment of autonomic regulation of the heart or cardiac beta-adrenoceptor desensitization.

Postnatal changes in the respiratory response of the conscious rat to serotonin 2A/2C receptor activation are reflected in the developmental pattern of fos expression in the brainstem

The influence on the breathing pattern of the activation of serotonin receptors belonging to the subtypes 2 A and 2 C (5-HT 2A/2C) has been assessed in newborn and adult conscious rats. Rats were given an acute intraperitoneal dose of the agonist DOI (1-(2.5-dimethoxy-4-iodophenyl)-2-aminopropane; 5 mg/kg). In newborns, DOI elicited a long-lasting respiratory depression by decreasing both tidal volume and respiratory frequency. In adults, DOI retained a depressant influence, although attenuated, on tidal volume. In contrast, it elicited an increase in respiratory frequency. In separate subsets of newborn and adult rats, immunohistochemistry has been used to monitor c-fos expression induced by DOI in the medullary and pontine regions involved in respiratory control. Counts of immunoreactive neurons indicated a marked increase in the neuronal populations activated in the adult compared to the newborn rat. The response to both experimental factors (newborn vs. adult controls) and drug (injected vs. control age-matched rats) were more pronounced in mature animals. Among developmental changes in the pattern of labeling, DOI elicited Fos expression in the adult but not in the neonate in the ventrolateral subnucleus of the nucleus of the solitary tract, the parabrachial area and the Kölliker-Fuse nucleus. This finding suggested that changes in the respiratory response to DOI might at least partly depend on maturational events within networks involved in the modulation of respiratory timing.

Circadian pattern of ventilation during acute and chronic hypercapnia in conscious adult rats

Because metabolism is a determinant of the ventilatory chemosensitivity, we tested the hypothesis that the ventilatory response to acute and prolonged hypercapnia is adjusted to the circadian oscillations in oxygen consumption (VO2). Adult rats were instrumented for measurements of body temperature (T(b)) and activity by telemetry. Pulmonary ventilation (VE) was measured by the barometric method and VO2 by the flow-through method. In the acute experiments, 16 conscious rats entrained to a 12:12-h light (L)-dark (D) cycle (lights on 7:00 AM) were exposed to air, 2%, and then 5% CO2 in normoxia (30-45 min each) at 11:00 AM and 11:00 PM. In a separate group of seven rats, simultaneous recordings of all variables were made continuously for 3 consecutive days in air followed by 3 days in 2% CO2 in normoxia, in a 12:12-h L-D cycle (lights on 7:00 AM). In air, all variables were significantly higher at night, whether rats were studied acutely or chronically. Acute CO2 exposure had similar significant effects at 11:00 AM and 11:00 PM on VE (approximately 25 and 100% increase with 2 and 5% CO2, respectively) and VO2 (approximately 8% drop with 5% CO2), such that the hyperventilatory response (% increase in VE/VO2 from air) was similar at both times. Chronic CO2 breathing increased VE at all times of the day, but less so during the L phase (approximately 15 vs. 22% increase in L and D, respectively), when activity was lower. However, VO2 was reduced from the air level (approximately 10% drop) in the L, such that the VE/VO2 response was similar between L and D. The same result was obtained when the VE/VO2 response was compared between the L and D phases for the same level of activity. These results suggest that, throughout the day, the hypercapnic hyperpnea, whether during acute or prolonged CO2, is perfectly adjusted to the metabolic level.

The circadian pattern of breathing in conscious adult rats

Recently, a circadian oscillation in pulmonary ventilation ( V ̇ e) was reported in conscious, undisturbed rats [Respir. Physiol. 120 (2000) 179], with a pattern similar to that of body temperature (Tb), oxygen consumption ( V ̇ O 2 ) and activity. The present study explored the relationship between the daily V ̇ e pattern and these rhythms. Adult rats ( n=23) were instrumented for measurements of Tb and activity by telemetry, and placed in a chamber for measurement of V ̇ e by the barometric method and V ̇ O 2 by an open-flow method. Simultaneous recordings were made continuously for 3 consecutive days in a 12-h light:12-h dark (L:D) cycle. All variables showed substantial daily oscillations, with significantly higher values in the D phase, and approximately proportionate changes in V ̇ e and V ̇ O 2 . Daily changes in tidal volume (V t) relative to inspiratory time (T i), rather than in T i relative to total breath duration, accounted for the oscillation in V ̇ e. The V t rhythm was phase-advanced relative to those of V ̇ O 2 and Tb. L–D differences in V ̇ e persisted when comparison between the phases was made for the same level of either very low or very high activity. We conclude that the oscillation in V ̇ e does not depend on the daily changes in activity. Rather, the daily pattern of V ̇ e is likely shaped by the oscillations of multiple physiological variables, two of which may be Tb and V ̇ O 2 .

Brainstem and hypothalamic areas activated by tissue hypoxia: Fos-like immunoreactivity induced by carbon monoxide inhalation in the rat

Acute ambient hypoxia interacts with the ventilatory and cardiocirculatory control systems, via the concomitant activation of arterial chemoreceptors and tissue oxygen-sensing mechanisms. Whether these latter mechanisms may trigger a specific pathway had not yet been elucidated. We addressed this issue, mapping Fos expression in adult conscious rats subjected to tissue hypoxia elicited by carbon monoxide inhalation, under conditions of minimal activation of arterial chemoreceptors. Brief stimuli have been delivered (1% carbon monoxide inhaled during 5, 10 or 20 min) to produce steady tissue hypoxia. Compared to normoxia, even the briefest stimuli led to marked neuronal activation within areas involved in ventilatory and cardiocirculatory control. In the brainstem, stimulated rats exhibited enhanced Fos expression in the nucleus of the solitary tract, the area postrema, the dorsal motor nucleus of the vagus nerve, the ventrolateral medulla, the parapyramidal group, the nucleus raphe pallidus, the lateral paragigantocellular nucleus, the locus coeruleus, the dorsal raphe nucleus, the lateral parabrachial area, and the ventrolateral central gray. In the hypothalamus, activated neurons were identified at the ventral border and in the supramamillary, posterior, and dorsomedial nuclei. Fos expression appeared with increasing the severity of tissue hypoxia in the retrotrapezoid nucleus, the ventral tegmental area and the arcuate and paraventricular hypothalamic nuclei. The present data support the idea that inputs related to tissue hypoxia might play a crucial role in patterning the physiological response to hypoxia.