Effects of age and clustered hypoxia on [(125)I] substance P binding to neurotachykinin-1 receptors in brainstem of developing swine.

This work focused on the postnatal development of substance P-bound neurotachykinin-1 (NK-1) receptors in the porcine brainstem using 2-3-, 6-11-, 16-18-, and 21-28-day-old piglets versus adult, and on alterations in these receptors after single and six-daily repeated clustered hypoxia using 6-11- and 21-28-day-old piglets. NK-1 receptor localization and densities were determined by quantitative autoradiography using mono-iodinated Bolton-Hunter substance P ([(125)I]BHSP). Slide-mounted brainstem sections, incubated in [(125)I]BHSP and then exposed to film, have shown [(125)I]BHSP binding throughout many brainstem nuclei and tracts, including the ambigual/periambigual (nAmb), dorsal motor vagal (dmnv), gigantocellular (nGC), hypoglossal (nHyp), medial parabrachial (nPBM), lateral reticular (nRL), raphe magnus (nRMg), raphe obscurus (nROb) and solitary tract (nTS) nuclei. NK-1 receptor densities decreased with age. As compared to normoxia, NK-1 receptor densities increased significantly after the six-daily hypoxia protocol in nAmb, dmnv, nHyp, nRL, nRMg, nROb, and nTS of both the young and older age groups. This increase may represent receptor upregulation as an adaptation to repeated hypoxia.

Respiratory responses to single and episodic hypoxia during development: mechanisms of adaptation.

The respiratory responses of the developmental subject to single and repeated episodes of hypoxia are distinct. During a single exposure, the fetus responds with an arrest of breathing activity, and the neonate, with excitation followed by depression (the biphasic response). Mechanisms under active consideration include chemosensory resetting, hypometabolism, prevalence of inhibitory neurotransmitter/modulator influence, and supramedullary regulation of control functions. When exposed to recurrent episodic hypoxia, neonates respond with relative hypoventilation, i.e. tolerance to a subsequent hypoxic stimulus. Whereas the investigation of processes responsible for this tolerance is at its infancy, studies using chronic hypoxia appear to be a useful guide. So far, altered interstitial neuromodulator levels and central markers of programmed neuronal death are harbingers of future research in this field. The clarification of the mechanisms involved in response to recurrent episodic hypoxia during development will be of fundamental value and may be useful for the eventual treatment and/or prevention of harmful central respiratory-related processes.

Repeated prenatal cocaine increases met-enkephalin immunoreactivity in respiratory-related medulla of developing swine.

Repeated prenatal exposure to cocaine is associated with attenuated respiratory and arousal responses in infants and piglets. As the normal development of these functions is influenced by medullary opioid systems, the present study explored the possible contribution of the opioid systems to the attenuation induced by cocaine. Methionine-enkephalin (met-enkephalin), an endogenous opioid peptide, was delineated by immunocytochemistry in respiratory- and arousal-related medullary regions of relatively immature (6-7-day-old) and more mature piglets (20-21-day-old). The animals were either unexposed, or exposed prenatally to 2 mg/kg cocaine four times daily administered to the pregnant sows intravenously throughout the last third of gestation. At control, met-enkephalin was found in the neurons, fibers and terminals of the respiratory- and arousal-related medullary regions throughout the age range studied. Prenatal cocaine exposure increased met-enkephalin immunoreactivity in the respiratory-related hypoglossal and solitary tract nuclei of both age groups. These findings support a modulatory role of met-enkephalin in the normal development of respiratory control, and an involvement of this peptide in the attenuation of respiration by repeated prenatal exposure to cocaine.

Prenatal cocaine raises mu-opioid receptor density in piglet cardiorespiratory medulla.

Repeated prenatal exposure to cocaine attenuates arousal and cardiorespiratory functions in neonates. This study explored the possible role of brainstem mu- and delta-opioid systems in these effects. Medullary sections were obtained from 6 to 7 (young) and 20 to 21-day-old (older) piglets either unexposed or exposed prenatally to a 2-mg/kg intravenous cocaine hydrochloride dose, injected to the pregnant sow four times a day during the last third of gestation. Mu- and delta-opioid receptor binding was assessed by quantitative autoradiography using, respectively, 125I-DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol) and 125I-DPDPE (Tyr-D-Pen-Gly-pCl-Phe-D-Pen). At control, delta-, but not mu-opioid, receptor density increased with postnatal age. In contrast, cocaine-induced mu-, but not delta-opioid, receptor density increased 1) in the dorsal motor vagal (dmnX) and facial (nF) nuclei, and, at borderline significance level, in the cardiorespiratory-related gigantocellular reticular nucleus (nRG) of the young, and 2) in the spinal trigeminal nucleus and tract (nSp5), and in the cardiorespiratory-related medial solitary tract (nTSm) and lateral reticular (nRL) nuclei of both age groups. These findings support a possible participation of the mu-opioid system in the attenuation of arousal and cardiorespiration after repeated prenatal exposure to cocaine.

Mu- and delta-opioid receptor densities in respiratory-related brainstem regions of neonatal swine.

The piglet displays similar postnatal development in respiration and sleep-wake behavior to the human. To shed light on the possible influence of opioid systems on these functions, this study assessed the density of mu- and delta-opioid receptors in brainstems of 2-3 and 5-7 (young), 14-17 (intermediate) and 20-21 (older) day-old piglets, using quantitative autoradiography. Serial 10 microns sections from fresh-frozen brains were incubated with either mu-(125I-DAGO) or delta-(125I-DPDPE) opioid ligands. The binding characteristics of each receptor remained unchanged over the age-range studied. delta-opioid receptor density was minimal in the young piglets, and increased over the age-range studied in all brainstem regions. mu-opioid receptor density exceeded delta-opioid density in all brainstem regions in young and older piglets, and remained unchanged with age. We conclude that, as in other species, the development of delta-opioid receptors in swine lags behind that of mu-opioid receptors, and that the distribution of each in the piglet's brainstem is distinct. The present findings help explain the changing influence of the mu- and delta-opioid systems on breating and state during postnatal development.

Prenatal cocaine alters normoxic sleep-wake and diaphragmatic EMG patterns in piglets.

This study assessed in piglets the effects of prenatal cocaine administration on sleep-wake states (SWS) and respiratory parameters, utilizing diaphragmatic electromyogram (EMGdi) recordings during normoxia before and after hypoxia (0.10 F(I,O2), 10 min). We asked whether the respiratory effects were linked to a specific SWS, and whether there was a difference in respiratory measures between the two normoxic conditions. Unsedated, chronically instrumented 3-9- or 21-31-day-old piglets, representing distinct stages in developmental respiratory control, were used. In pre-hypoxic normoxia, prenatal cocaine enhanced sleep at the expense of wakefulness and increased EMGdi amplitude, slope, and area in both age groups regardless of SWS; after the hypoxia, the respiratory findings persisted in the young group, but disappeared in the older group [corrected]. In the young group and regardless of SWS, interbreath interval (ttot) and expiratory duration (ttot - tEMGdi[duration of EMGdi]) were shorter in the cocaine-exposed than in the unexposed piglets, and ttot, tEMGdi, and (ttot - tEMGdi) decreased from pre- to post-hypoxic normoxia. In the older group, ttot and (ttot - tEMGdi) differed among SWS, but were unaffected by drug treatment; tEMGdi was higher with cocaine exposure in pre-, but not in post-hypoxic normoxia, and two-thirds of the EMGdi measurements during post-hypoxic normoxia exhibited a similar magnitude in the drug-treated and untreated groups regardless of SWS. We conclude that 1) prenatal cocaine alters both SWS and EMGdi, but the EMGdi effects are independent of SWS; and 2) the similar EMGdi patterns in the older group after hypoxia, regardless of drug treatment, suggest that hypoxia and chronic prenatal cocaine might alter EMGdi by similar mechanisms.

Curtailed respiration by repeated vs. isolated hypoxia in maturing piglets is unrelated to NTS ME or SP levels.

In early development, respiratory disorders can produce recurring hypoxic episodes during sleep. To examine possible effects of daily repeated vs. isolated hypoxic hypoxia, cardiorespiratory functions and central, respiratory-related neuromodulator levels in 21- to 32-day-old, chronically instrumented, unsedated piglets were compared between a fifth sequential daily hypoxia and an isolated hypoxia (10% O2-90% N2 for 30 min). Diaphragmatic electromyographic activity, heart rate and arterial pressure, and pH and gas tensions were measured. In vivo microdialysis, via chronically implanted guides, served to sample interstitial substance P (SP) and methionine-enkephalin (ME) at the level of the respiratory-related nucleus tractus solitarii (NTS). Compared with an isolated hypoxia, repeated hypoxia resulted in 1) lower respiratory frequency (f), ventilation equivalent, and arterial pH, higher arterial PO2 during hypoxia, and lower f in recovery from hypoxia; and 2) increased SP concentrations but no change in ME concentrations. We conclude that, in these maturing swine, repeated vs. isolated hypoxic exposure curtails respiratory responses to hypoxia by a mechanism(s) unrelated to SP or ME levels at the NTS.

Repeated microdialysis from the nucleus tractus solitarii of chronically instrumented, unsedated piglets.

Normal development of respiratory rhythm and control, and perturbations thereof, likely relate to neuromodulators in brainstem regions. To assess the feasibility of repeated neurochemical sampling by in vivo microdialysis from the respiratory-related nucleus tractus solitarii (NTS) during development. 19-24 day-old piglets (n = 7) were implanted under anesthesia with chronic microdialysis guides near NTS around obex. Unsedated piglets then underwent in vivo microdialysis twice, 3 days apart, through probes inserted acutely via the guides to abut against the NTS. The probe tips, surrounded by normal neurons and only diffuse gliosis, either intersected, or were within < or = 300 microns from the NTS. Thirty-min microdialysates were collected for 120 min in normoxia, HPLC-fractionated, and assayed for substance-P (SP), a respiratory excitatory neuropeptide. SP levels stabilized within 90 min from probe placement, and did not differ between the 2 experimental days. Thus, repeated in vivo microdialysis from NTS of conscious piglets is feasible, and can illuminate respiratory-related normal and pathological neurochemical processes during development.

Respiratory responses to rapid-onset, repetitive vs. continuous hypoxia in piglets.

Repeated, frequent hypoxic exposures may precede Sudden Infant Death. This study assessed whether such hypoxic modality, vs. continuous hypoxia, compromised compensatory cardiorespiratory responses. Following aseptic, chronic instrumentation, 10 to 20 day-old, unsedated piglets underwent measurements of arterial O2 saturation, pH and gas tensions, respiration, heart rate, physical activity, O2 consumption and rectal temperature on several experimental days. The piglets were exposed to 21 min of either 10% or 6% O2 in N2, each comprising either seven, 3-min exposures alternating with 3-min intervals in 21% O2 balance N2, or 7 consecutive hypoxic exposures. Responses to 6% hypoxia were greater than those to 10% hypoxia. In 10% hypoxia, responses to repetitive vs. continuous exposure differed only in PaCO2. In 6% hypoxia, repetitive vs. continuous exposure resulted in lower respiratory frequency (p < 0.05) and in lower ventilation equivalent (p = 0.07) despite higher activity levels. Thus, the mode of hypoxic exposure determines the extent of the respiratory response: Severe, repetitive hypoxia mitigates protective respiratory responses when compared to equivalent, but sustained hypoxia.

Electrocorticographic activity during repeated vs continuous hypoxia in piglets.

To assess the effects on brain activity of repeated vs continuous hypoxia, 16, 10- to 22-day-old piglets were instrumented chronically for electrocortical and arterial pH and gas tension measurements. They inhaled 10% or 6% O2 in N2 for 21 min, either continuously, or during seven, 3 min exposures interrupted by 3 min recovery periods in air, all while behaving naturally within a sealed, temperature controlled, plexiglass box. An isoelectric electrocorticogram (ECoG) and/or seizures, related to the onset of hypoxia, occurred repeatedly in 6 of the 10 exposures to 6% repetitive hypoxia, only twice in 8 exposures to 6% continuous hypoxia, and never in 10% hypoxia. A frequency analysis of the ECoG, excluding all sections exhibiting isoelectric and seizure activity, revealed no changes with 10% hypoxia, but a shift towards the lower bands during both repetitive and continuous 6% hypoxia. The extent of these shifts was greater in records that also displayed isoelectric ECoG and/or seizures. The ECoG spectrum recovered at the end of the hypoxic exposure, but not when isoelectric ECoG and/or seizures coexisted. We conclude that repeated, frequent episodes of hypoxia are more detrimental than a prolonged single event, and may contribute to the occurrence of Sudden Infant Death.

Prenatal cocaine alters diaphragmatic EMG responses to hypoxia in developing swine.

To distinguish pure effects of prenatal cocaine exposure on respiratory control from confounding factors inherent to drug abuse, a porcine model was established. Cocaine was administered at 2 mg/kg 4 times daily during 0.66-1.0 gestation to 5 paired sows. At birth, cocaine-exposed piglets were fostered to the unexposed paired sows and their litters. Respiratory measures were obtained from diaphragmatic electromyographic activity (EMGDI) of 3 to 9 (young) and 21 to 31 (older) day-old, chronically instrumented piglets during 10 min each of normoxia and hypoxia (10% O2 in 90% N2), and compared between cocaine-exposed and unexposed animals. Arterial pH and gas tensions in hypoxia were not altered by cocaine. In the young neonates, only during hypoxia, cocaine preexposure produced a transient elevation of the peak and initial slope of the integrated EMGDI envelope, but did not affect respiratory timing, provided no extensive periodic breathing or apnea had occurred. In the older animals, during hypoxia only, cocaine preexposure increased the peak and initial slope of EMGDI envelope while decreasing summed EMGDI activity, EMGDI duration and Ttot toward levels seen in the young unexposed neonates. These findings suggest that prenatal exposure to cocaine retards the normal maturation of respiratory EMG responses to hypoxia.

Microdialyzed adenosine in nucleus tractus solitarii and ventilatory response to hypoxia in piglets.

Levels of adenosine, inosine, and hypoxanthine from the interstitial space at the nucleus tractus solitarii were measured by microdialysis in eight 20- to 25-day-old anesthetized spontaneously breathing piglets. Microdialyzed samples were collected every 30 min for 2 h after the insertion of the probe to ensure stability of purine levels and then during 30 min each of normoxia, hypoxia (10% O2-90% N2), and normoxia. The purines were separated by high-pressure liquid chromatography with ultraviolet detection and quantified at 254-nm wavelength. Tidal volume, breathing frequency, minute ventilation, mean arterial blood pressure, pH, and gas tensions were measured. Compared with control, adenosine levels during hypoxia increased by 40.7 +/- 5.5% and then tended to decline during the recovery from hypoxia, but the levels remained higher than in control. Ventilatory measures exhibited a modest biphasic pattern during hypoxia and resumed control values by 10 min after the removal of the hypoxia. The increased adenosine release during hypoxia provides additional evidence for the possible participation of adenosine in the central suppression of breathing during hypoxia.

Chronic prenatal cocaine retards maturation of state and of respiratory patterns in swine.

This study assessed effects of prolonged prenatal cocaine exposure on respiratory pattern and sleep-wake states in a postnatal porcine model. Yucatan miniature sows received 2 mg/kg cocaine intravenously four times daily during 0.66-1.0 gestation. At birth, cocaine-exposed litters were fostered to unexposed paired sows and their litters. Chronically instrumented piglets were studied at 3-9 (young) and 21-31 days (older). Sleep-wake states were determined from electrocorticogram, eye movements, submental electromyogram, and behavior, and respiratory patterns were determined from diaphragmatic and posterior cricoarytenoid electromyograms (EMGdi and EMGpca, respectively). Under baseline conditions, prenatal cocaine 1) increased the number of apneas expressed by silence of EMGdi or EMGpca and prolonged the duration of EMGpca-related apneas at both ages; 2) increased the number of periodic breathing episodes at both ages; 3) increased percent time of active sleep and decreased that of wakefulness at both ages; and 4) increased time in quiet sleep in the older animals, producing in them a sleep-wake distribution similar to that of the young neonates. Whereas the findings in the youngest piglets may have been influenced by persistent systemic cocaine, those in the older preexposed piglets, devoid of systemic cocaine, imply that chronic prenatal cocaine retards the postnatal maturation of state and respiratory pattern.

Cardiorespiratory and sleep-wake behavior in developing swine: kappa-opioid influence.

Effects of specific kappa-opioid antagonism with norbinaltorphimine (NorBNI) on sleep-wake state, blood pressure and heart rate, and on diaphragmatic and posterior cricoarytenoid electromyographic activities were assessed in 3 to 13 and 23 to 33 day-old, chronically instrumented, unanesthetized piglets. Preliminary experiments established the pharmacodynamics and dose-response for NorBNI. In the main study, each piglet was studied twice daily, once before and once after 3.7 mg kg-1 NorBNI iv, for up to five consecutive days. During each study session, piglets underwent 10 min trials with 21% O2 in 79% N2 followed by 10% O2 in 90% N2 while lying in a sling within a plexiglass box. Sleep-wake distribution and cardiorespiratory functions matured with age. NorBNI produced a modest increase of arterial pressure and heart rate in the older group only, and altered neither state nor respiration at either age. These results suggest that, in the developing piglet model, the kappa-opioid system influences neither breathing nor state, but modulates cardiovascular regulation to a modest degree and later in ontogeny.

Age-related mu-, delta-and kappa-opioid ligands in respiratory-related brain regions of piglets: effect of prenatal cocaine.

Neonates, as compared to older subjects, exhibit increased signs of relative respiratory suppression such as apnea, periodic breathing and only transient hyperventilatory response to hypoxia. Prenatal cocaine exposure exaggerates the respiratory pattern disturbances observed in infants. As endogenous opioids cause central suppression of breathing, we tested their possible involvement in these effects by assessing opioid content in respiratory-related brainstem regions of 2 to 5 (young) and 18 to 22 (older) day-old piglets, unexposed or preexposed to cocaine during 0.66 to 1.0 gestation. The selected ages represent distinct stages in the postnatal development of respiration. beta-Endorphin, methionine-enkephalin, dynorphin A and dynorphin B from the tractus solitarii, ambigualis, gigantoreticularis and parabrachialis medialis nuclei were separated by high performance liquid chromatography, then quantified by radioimmunoassays. Opioid content was higher in the brain regions of the young than of the older piglets, and increased after cocaine exposure in both age groups, but more in the young. These findings support the possible contribution of high opioid content to the relative suppression of respiratory function in early life, and to the exaggerated respiratory dysrhythmia observed in cocaine preexposed neonates.

Met-enkephalin-like immunoreactivity in microdialysates from nucleus tractus solitarii in piglets during normoxia and hypoxia.

Met-enkephalin-like immunoreactivity in microdialysates from the respiratory-related nucleus tractus solitarii was determined simultaneously with ventilatory responses in seven, spontaneously breathing, developing swine under conditions of normoxia, hypoxia and recovery from hypoxia for 30 min each. Assayed levels of Met-enkephalin-like immunoreactivity in normoxia were 0.89 +/- 0.23 pg/microliters. These levels increased to 203.6 +/- 32.2% and 283.1 +/- 55.8% of control during hypoxia and recovery, respectively. Hyperventilation during hypoxia was not sustained, comprising brief stimulation followed by return to near-control level. Taken together, these results provide further evidence that opioid release may contribute to the suppression of ventilation in hypoxia during development.

Prenatal cocaine alters open-field behavior in young swine.

Yucatan minisows received 2 mg/kg cocaine i.v. 4 times daily during the last third of gestation. Their piglets were fostered at birth to paired, unexposed sows with their litters, and studied at age 2 to 9 (young group) and 22 to 29 days (older group). Three to 5 exposed and unexposed piglets of each age group were videotaped together for 30 min on 5 consecutive days in an open-field environment. For each piglet, 41 behaviors were scored, timed, summed and clustered into 9 behavioral categories. With age, and independently of drug exposure, piglets spent more time in ingestion, immobility while alone and play/aggression, and less time in group locomotion. For the first 4 test days, the young exposed piglets spent more time in group immobility and less time in individual locomotion and rooting than their age-matched controls. In contrast, the older exposed and unexposed piglet groups did not differ in any of these behavioral clusters. These results suggest that prenatal cocaine exposure in neonatal swine may transiently affect responses to spatial novelty.

Age-related electrocorticographic and respiratory adaptation to repeated hypoxia.

Severe hypoxia is known to produce depression in electrical brain activity and perturbation of respiratory pattern. In piglets undergoing chronic recording of brain and respiratory muscle activities, a depressed electrocorticogram (ECoG) was observed in response to rapidly induced (< 30 s), brief (10 min), and moderate hypoxia (10% O2 in 90% N2) in 16 out of 42 study sessions in young (3- to 11-day-old) animals only. Responses to hypoxia were monitored over 4 consecutive days. In five cases, the latency to the onset of the ECoG depression increased progressively over the 4 test days, and its duration decreased progressively. An associated respiratory gasping pattern also exhibited gradual remission over consecutive days. These changes in the responses to repeated hypoxia demonstrate adaptation of mechanisms underlying neuronal perturbation by oxygen deprivation.

Hypoxia, sleep and respiration in relation to opioids in developing swine.

To test the role of mu and delta opioid systems in neonates during hypoxia, a total of sixteen, 4-11 (n = 7) and 26-33-day-old piglets (n = 9) were instrumented aseptically for assessment of sleep/wake states (S/W), electromyographic activities of the diaphragm and posterior cricoarytenoid muscles (EMGdi, EMG-pca, respectively), heart rate, and arterial pressures, pH and gas tensions. During daily sessions for 5 consecutive days, the piglets inhaled 10% O2/90% N2 for 10 min twice per session, first before any drug, then after either naltrexone (2 mg.kg-1 i.v.), a predominantly mu opioid antagonist, or naltrindole (4 mg.kg-1 i.v.), a specific delta opioid antagonist. During hypoxia, young, in contrast to older piglets, spent more time asleep, and increased sleep during the second half of the hypoxic exposure before, but not after each antagonist. They also exhibited, overall, higher breathing frequency, and lower slope, amplitude, area and initial area of EMGdi and EMGpca activity than older piglets. Naltrindole stimulated EMGpca activity in both age groups, and naltrexone increased the breathing frequency and slope of EMGdi in the older group. We conclude that hypoxia enhances the activation of central mu and delta opioid systems which influence S/W and respiration.

Respiratory electromyographic estimates of ventilatory functions in piglets.

The best electromyographic (EMG) predictors of respiratory drive (P100), tidal volume (VT) and ventilation (VE) were determined from diaphragmatic (DI) and posterior cricoarytenoid (PCA) EMG measures in 8-48-day-old, anesthetized piglets. Progressive hypercapnia was employed to obtain a wide range of muscle activity. A custom-designed, microcomputer-based system was employed to measure the duration, peak amplitude, rate of rise (initial slope) as well as the summed total and initial (first 100 ms) EMG activity from the DI and the PCA. For each respiratory function, the following combinations of EMG measures were identified as significant predictors using regression analyses: (1) for P100, DI amplitude, PCA initial area and PCA rate of rise; (2) for VT, DI amplitude, PCA duration and DI duration; (3) for VE, DI amplitude, DI initial area, PCA initial area, PCA rate of rise, PCA duration, DI area and DI rate of rise. Thus, whereas the traditionally employed measure of DI amplitude is an important correlate of P100, VT or VE, a complete estimate of these respiratory functions requires the inclusion of initial EMG measures and duration.