A. H. Jansen

Sleep, wakefulness and the monosynaptic reflex in fetal and newborn lambs

Abstract1.Electrocorticogram (ECoG), electromyogram (EMG) of the lateral rectus and antigravity muscles (neck and masseter) and breathing activity (FB) were monitored in chronically prepared fetal sheep of 125–140 days gestation and in newborn lambs up to 11 days postnatal age.2.Awake state (AW), non-rapid eye movement sleep (NREM) and rapid eye movement sleep (REM) were defined using standard criteria for ECoG, eye movements and postural muscle tone.3.The percentage of time the fetuses spent in each state was: NREM sleep 53%, REM sleep 41.4%, and AW 5.6%.4.Spontaneous intrauterine breathing activity occurred only during REM sleep, but 35% of REM sleep was not associated with FB.5.Gasps (isolated deep inspirations) appeared occasionally throughout the recording and were not related to any specific sleep state or wakefulness.6.In the fetus, the monosynaptic reflex (MSR) induced by direct electrical stimulation of the fibular nerve was enhanced by about 75% during REM sleep compared to NREM and AW. In the newborn lamb the adult pattern of suppression of MSR during REM sleep was not seen until several days after birth.

Influence of sleep state on the response to hypercapnia in fetal lambs

The effect of sleep state on the respiratory response to hypercapnia was studied in 14 chronic fetal sheep, 125-140 days gestation. Fetal PaCO2 was raised by 11 to 34 Torr by gradually increasing the maternal FICO2 to 0.09. Fetal sleep state was monitored. Fetal breathing (FB) was analysed in terms of frequency (f), tracheal pressure (TP) and ventilation equivalent (VEq) = sigma TP /min. In 16 out of 17 experiments on apneic fetuses in NREM sleep, the fetuses switched to REM sleep and in 14 instances began to breathe within 2 1/2 min thereafter. The PaCO2 at which apneic fetuses started breathing was 54.8 +/- 8.4 Torr (mean +/- SD). In 4 out of 10 trials on breathing fetuses in REM sleep the fetuses switched to NREM sleep and stopped breathing before removal of the CO2 stimulus. During REM sleep hypercapnia stimulated FB by an increase in TP and by a reduction in the number and duration of apneic pauses. It is concluded that in the fetal lamb CO2 stimulates breathing only during REM sleep and that this stimulus is superimposed on the basic mechanism that stimulated spontaneous FB during this sleep state.

Contribution of the adrenal glands and splenic nerve to LPS-induced splenic cytokine production in the rat.

Both the hypothalamic pituitary adrenal axis (HPAA) and the sympathetic nervous system (SNS) can inhibit immune function and are regarded as the primary efferent pathways for neural-immune interactions. To determine if this relationship is maintained in vivo in response to an inflammatory stimulus, rats were injected intravenously (iv) with various doses of lipopolysaccharide (LPS) and splenic cytokine mRNA and protein levels were measured at several dose and time intervals post-injection. The spleen was chosen as the target organ because both the neural and hormonal inputs to the spleen can be selectively removed by splenic nerve cut (SNC) and adrenalectomy (ADX), respectively. Data from our dose response studies established that maximum levels of splenic cytokines were induced in response to relatively low doses of LPS. Minimal changes in LPS-induced splenic cytokine levels were observed in response to ADX, SNC, or a combination of the two procedures across several doses of LPS. These results suggest that there are aspects of immune regulation that are functionally removed from these central modulatory systems and that the counter-regulatory responses induced by LPS have minimal impact on the concurrent induction of cytokines by this inflammatory stimulus. The conceptual model of neural-immune regulation as an inhibitory feedback system, at least with regards to the early activational effects induced by an inflammatory stimulus, was not supported by these studies.

Respiratory response to somatic stimulation in fetal lambs during sleep and wakefulness

Abstract1.The respiratory response to electrical stimulation of the nose, tail, gum and fibular nerve (nonspecific somatic stimulation) was tested in chronically instrumented fetal lambs during sleep and wakefulness.2.The respiratory response to somatic stimulation was greatest during REM sleep, lowest during NREM sleep, and intermediate during the awake state (AW).3.Respiratory responses could follow electrical somatic stimulation up to 2 Hz in any sleep state without changing sleep state.4.The fetal breathing (FB) response to repetitive stimulation depended on the initial sleep state and whether or not the sleep state was affected by the stimulation. Five patterns of response were seen:a)NREM→NREM (initial sleep state and sleep state at end of stimulation) — FB was initated and did not continue beyond the duration of the stimulation.b)NREM → REM sleep — FB was initiated and continued beyond the time of stimulation.c)REM → REM sleep — spontaneous FB was enhanced and continued beyond the time of stimulation.d)REM → NREM sleep — spontaneous FB was enhanced but did not continue beyond the time of stimulation.e)NREM, REM sleep, Awake → Awake — FB was initiated or enhanced and continued beyond the time of stimulation until the onset of NREM sleep.

Effect of sinus denervation and vagotomy on c-fos expression in the nucleus tractus solitarius after exposure to CO2.

Exposure to hypercapnia and electrical stimulation of the carotid sinus nerve (CSN) has been shown to induce c -fos expression in several brain stem regions including the nucleus tractus solitarius (NTS). To test whether the labeled neurons were activated directly by hypercapnia or secondarily via the carotid bodies (sinus nerve), adult rats were exposed to either air or 14-16% CO2 for 1 h. Experiments were done on eight groups: (1) exposure to air, (2) exposure to CO2, (3) chronic CSN denervation/CO2, (4) chronic unilateral CSN denervation/CO2, (5) chronic sham CSN denervation/CO2, (6) anesthetized/CO2, (7) anesthetized and acute vagotomy /CO2, and (8) premedicated with morphine, 10 mg s.c, 20 min before exposure to CO2. After exposure to CO2 or air the rats were anesthetized, perfused with 4% paraformaldehyde and the brains processed for immunohistochemical staining for c -fos protein using the PAP (i.e. peroxidase anti-peroxidase) technique. Labeled neurons in the area of the NTS in every second 50-µm section were counted and their position plotted using a microscope and camera lucida attachment. Rats exposed to CO2 had a significantly greater number of labeled neurons in the NTS than those exposed to air. Other interventions, such as CSN denervation, surgery, anesthesia, vagotomy or injection of morphine did not significantly affect the level of c -fos expression in rats exposed to hypercapnia, indicative of central stimulation rather than secondary peripheral input. These responsive neurons may be part of a widespread central chemoreceptive complex.

Influence of prolonged adenosine receptor blockade on fetal sleep and breathing patterns

Chronically prepared fetal sheep were subjected to 48 h infusions of theophylline, an adenosine antagonist, enprofylline, a xanthine without adenosine antagonism, or saline. Theophylline increased mean (+/- SD) incidence of REM sleep from 49.3 +/- 8.3% to 57.3 +/- 6.7% (p < 0.02) and wakefulness from 1.3 +/- 1.4% to 8.1 +/- 7.1% (p < 0.01). On the first day of theophylline infusion incidence of fetal breathing (FB) increased from 37.9 +/- 8.1% to 53.7 +/- 11.6% of total time (p < 0.002) and from 76.4 +/- 10.2% to 87.6 +/- 10.3% of REM sleep (p < 0.02). Diaphragmatic EMG/min increased from 6.9 +/- 4.0 to 17.3 +/- 13 arbitrary units (p < 0.02). By the second day of infusion, FB had returned to baseline value. Enprofylline and saline had no effect. 125 micrograms phenyl isopropyl adenosine (PIA) i.v. caused fetal apnea that was reduced from 143 +/- 45.5 min on the control day to 39.8 +/- 34.7 min (p < 0.001) during theophylline infusion. Enprofylline and saline had no effect, suggesting that the observed theophylline effect was due to its adenosine antagonism rather than to non-specific xanthine action. We conclude that endogenous adenosine suppresses FB, but since theophylline did not alter the basic relationship between FB and REM sleep it is not primarily responsible for apnea during NREM sleep.

Influence of naloxone on fetal breathing and the respiratory response to hypercapnia

The effect of naloxone on fetal breathing and the respiratory sensitivity to CO2 was tested on chronically prepared fetal lambs on days one and four post-surgery. After a control period the fetus was challenged with hypercapnia for 10 min and after another control period 9 mg naloxone was administered to the fetus followed by another CO2 test 15 min later. An index of fetal breathing (Veq), tidal volume (VT) and frequency of breathing (f) was determined from tracheal pressure deflections and from the integrated diaphragmatic EMG, expressed as power of diaphragmatic activity per min. Naloxone consistently caused fetal arousal but the duration was variable. The respiratory response to naloxone was also variable and not statistically different from control. The respiratory sensitivity to CO2 (% delta Veq/Torr delta PaCO2 or % delta Diaph. Power/min/Torr delta PaCO2) was not changed by naloxone on either day. We conclude that endorphins do not have a significant direct role in the fetal respiratory response to CO2 but may be involved in the control of state.

Neuropeptide specificity of prostaglandin E2-induced activation of splenic and renal sympathetic nerves in the rat

Sympathetic activation occurs rapidly following intracerebroventricular (icv) injection of prostaglandin E2(PGE2). This study examined whether neuropeptides mediate PGE2-induced sympathetic nerve activation in urethane/chloralose-anesthetized Sprague-Dawley rats. Animals were pretreated (20.0 microg, icv) with the following receptor antagonists; CRF ([D-Phe12,Nle21,38,Calpha-MeLeu37]CRF12-41), AVP-V1 (Des-Gly-[Phaa1, D-Tyr(Et)2,Lys6,Arg8]-vasopressin), or OT (OT+V1, [d(CH2)5,Tyr(Me)2,Orn8]-vasotocin) followed 20 min later by PGE2 (2.0 microg, icv). Pretreatment with the CRF antagonist attenuated the increase in renal nerve activity induced by PGE2 when measured 10 and 30 min post-injection. PGE2-induced renal nerve activity was also inhibited at both time points by the AVP antagonist and, to a similar extent, the OT antagonist. The AVP antagonist did not effect splenic nerve responses to PGE2 whereas the CRF antagonist produced an incomplete and transient reduction in PGE2-induced activation of the splenic nerve. However, the OT antagonist completely blocked the activation of the splenic nerve after central injection of PGE2. ICV injections of AVP and OT produced immediate changes in splenic and renal nerve activity whereas CRF failed to alter the activity of either nerve in anesthetized or conscious animals. Thus, PGE2 acts through neuropeptide-specific pathways to initiate sympathetic outflow and OT is a specific component of the sympathetic pathway innervating the spleen.