α-Electrophilic Reactivity of Nitronates.

Chemistry of covalent nitronates regarding nucleophilic addition to C=N bond is described. Various types of electrophilic activation of nitronates and stability of formed products are discussed with main attention paid to authors' work in the area.

Expression of the leptin receptor during germ cell development in the mouse testis.

Leptin, a recently identified hormonal product of the ob gene, is known to regulate appetite, body metabolism, and reproductive functions. We investigated the expression of the leptin receptor (Ob-R) in testes from different age groups. The messenger RNA for Ob-R was found in testes from all age groups using RT-PCR. Using immunohistochemistry, we observed age- and stage-dependent distribution of the Ob-R in mouse testis. In testis of 5-day-old mice, its expression was mainly in type A spermatogonia. In the 20- and 30-day-old testis, Ob-R expression was in the spermatocytes; in the adult testis, it was specific to spermatocytes in stages IX and X of the cycle of the seminiferous epithelium. Five main immunoreactive proteins were detected using Western blot (220, 120, 90, 66, and 46 kDa). The 120-kDa protein was evident only in 20-day-old and older testes, whereas the 90-kDa band was present only in the 5- and 10-day-old testis. Leptin treatment induced phosphorylation of signal transducer and activator of transcription-3 in cultured seminiferous tubules from adult and 5-day-old testes. Our results show for the first time age- and stage-specific localization of a functional Ob-R in testicular germ cells. We hypothesize a direct role for leptin, through phosphorylation of signal transducer and activator of transcription-3, in proliferation and differentiation of germ cells, which may partially explain the infertility observed in leptin-deficient mice.

Hypercapnia alters sleep state pattern.

Mild hypercapnia in the adult animal does not affect sleep pattern but more severe hypercapnia in the fetus increases the duration of REM sleep. Adult male rats were exposed daily for 2-3 h sessions at random to 6, 7, and 8% CO2 or room air. Breathing CO2 caused a 60% increase in sleep onset latency, a 28% decrease in sleep duration, but no change in percent time spent in REM. However, the duration of REM sleep episodes increased by 30%. Thus, in both fetal and adult animals severe hypercapnia appears to have a similar effect on sleep pattern.

Fetal respiratory neuronal activity during REM and NREM sleep.

Chronically prepared near-term fetal lambs (129-133 days gestation) were exteriorized into a saline bath under maternal spinal anesthesia, and each head was rigidly connected to a stereotaxic frame. Multibarrel glass electrodes were inserted into the region of the nucleus tractus solitarius (NTS) during fetal breathing (FB) in rapid-eye-movement (REM) sleep. Of a total of 223 neurons, it was possible to record only 6 neurons for which firing amplitude did not change during the transition from REM to non-REM (NREM) sleep. The burst frequency, number of spikes per breath, and association with diaphragmatic activity were variable, with phasic activity preceding FB or disappearing and reappearing during FB. During the transition from REM to NREM sleep, phasic neuronal activity ceased, became tonic, and finally ceased altogether. L-Glutamate increased the number of spikes per breath and caused previous phasic activity to reappear but in NREM sleep produced only tonic activity. We conclude that during REM sleep the fetal respiratory neurons in the region of the NTS are to a large degree influenced by nonrespiratory REM sleep factors and that quiescence of respiratory neurons during NREM sleep is due to the lack of phasic input rather than to direct inhibition. Inhibition of FB during NREM sleep must occur upstream of the NTS neuron.

Maturation of spontaneous fetal diaphragmatic activity and fetal response to hypercapnia and hypoxemia.

The electromyogram (EMG) of the diaphragm, lateral rectus, and nuchal and hindlimb muscles were studied during spontaneous activity and during hypercapnia or hypoxemia in eight fetal sheep from 0.5 to 0.8 gestation (73-128 days). At the earliest gestational age, diaphragmatic EMG activity was mainly tonic and associated with tonic activity of somatic muscles. The stimulus for the diaphragmatic activity originated centrally. Brief periods of a rapid-eye-movement (REM) state characterized by phasic lateral rectus and diaphragmatic activity and absence of nuchal activity were recognized. Furthermore, from 0.5 to 0.7 gestation onward, activity of all muscles increased. Thereafter increased specificity of activity in relation to the apparent REM and non-rapid-eye-movement (NREM) state occurred. With maturation, phasic diaphragmatic activity increased at the expense of tonic activity. The most striking effect of maturation on apnea was a greater proportion of apnea lasting greater than 1 min, but the total duration of apnea as a percent of a total recording remained unchanged. The quantitative response to hypercapnia during maturation was independent of the pattern of spontaneous diaphragmatic activity. Hypercapnia at 0.5 gestation changed the pattern of diaphragmatic EMG activity from mainly tonic to phasic. Thus the central chemoreceptors and appropriate neuronal pathways are present and functional as early as 0.5 gestation. Hypercapnia at 0.5 gestation caused a shift in diaphragmatic EMG power to lower frequencies similar to that found during control conditions in the older fetus. This might suggest that during maturation there is increased recruitment of phrenic motoneurons. Hypoxemia abolished tonic somatic activity at 0.5 gestation and decreased phasic diaphragmatic activity at more advanced gestational ages. Therefore the central inhibitory mechanisms of hypoxemia are developed by 0.5 gestation.

Stimulation of fetal breathing activity by beta-adrenergic mechanisms.

Experiments were done on chronically prepared fetal lambs, 125-135 days gestation, to test the effects of various catecholamines on fetal breathing (FB) as well as the influence of isoproterenol on the fetal respiratory response to hypoxemia. Bolus injections of epinephrine, norepinephrine, and isoproterenol (5-20 micrograms) were administered via the lingual artery or femoral or jugular vein during periods of FB activity or apnea. The effects of epinephrine and norepinephrine on FB were variable and not statistically significant. Isoproterenol produced a significant increase in FB, frequency of breathing, and mean inspiratory effort, when infused during rapid-eye-movement (REM) sleep but it failed to induce FB during non-rapid-eye-movement (NREM) sleep. The positive response during REM sleep was absent following pretreatment with 3-5 mg propranolol and after bilateral section of the sinus nerves. The effect of hypoxia on FB was tested before and during constant infusion of isoproterenol (1 microgram/min iv). A reduction of the fetal arterial PO2 by 3-10 Torr produced the characteristic depression of FB in either situation. These results indicate that the fetal carotid body chemoreceptors can reflexly stimulate FB under certain circumstances but that their effectiveness is limited by more powerful inhibitory mechanisms such as those operative during NREM sleep and hypoxemia.

Effects of hypercapnia and hypoxemia on fetal breathing after decortication.

The effects of hypercapnia and hypoxemia on breathing movements were studied in 12 chronically decorticated fetal sheep, 127-140 days gestation. The fetal state of consciousness was defined in terms of activity of the lateral rectus and nuchal muscles. Arterial blood pressure was monitored. Fetal breathing was determined by integrated diaphragmatic electromyogram (EMG) and analyzed in terms of inspiratory time (TI), expiratory time (TE), electrical equivalent of tidal volume (EVT), breath interval (TT), duty cycle (TI/TT), mean inspiratory flow equivalent (EVT/TI), and instantaneous ventilation equivalent (EVT/TT). Fetal breathing occurred only during episodes of rapid-eye movements, and the response to hypercapnia consisted of an increase in EVT, TI, EVE, and EVT/TI and a decrease in the coefficient of variation of all measured parameters. Induction of hypoxia during episodes of spontaneous fetal breathing produced a decrease in the rate of breathing and an increase in EVT and TI with no change in the variability of all parameters studied. Since similar responses to hypercapnia and hypoxemia are seen in the intact fetus, we conclude that the cerebral cortex has no obvious effect on the chemical control of fetal breathing.

Analysis of respiratory neuronal activity in fetal sheep.

We developed a new method to monitor fetal medullary respiratory neurons utilizing a two-stage approach. At 129-133 days of gestation, sheep were anesthetized, and a window was placed over the area of the fourth ventricle. After a recovery period of 3-5 days, the fetus was exteriorized into a saline bath under maternal spinal anesthesia, and the head was connected rigidly to a stereotaxic frame. Microelectrodes were inserted into the area of the nucleus tractus solitarius during rapid-eye-movement sleep, and extracellular recordings of 223 respiratory neurons were analyzed: 76% were inspiratory, 9% expiratory, and 15% phase spanning, as classified by visual and computer correlation to diaphragmatic activity. More detailed analysis of 100 neurons was done to assess the respiratory component (eta 2) by use of a modification of the method developed by Orem and Dick (J. Neurophysiol, 50: 1098-1107, 1983). With use of cohorts of 25 breaths, fetal respiratory neurons were found to frequently change their phase relationship to diaphragmatic activity. The eta 2 statistic of fetal respiratory neurons was not a stable characteristic but changed over time. This could be a reflection of an immature central respiratory system before birth or the lack of major sensory inputs.

Maturation of steady-state CO2 sensitivity in vagotomized anesthetized lambs.

The maturation of the respiratory sensitivity to CO2 was studied in three groups of anesthetized (ketamine, acepromazine) lambs 2-3, 14-16, and 21-22 days old. The lambs were tracheostomized, vagotomized, paralyzed, and ventilated with 100% O2. Phrenic nerve activity served as the measure of respiration. The lambs were hyperventilated to apneic threshold, and end-tidal PCO2 was raised in 0.5% steps for 5-7 min each to a maximum 7-8% and then decreased in similar steps to apneic threshold. The sinus nerves were cut, and the CO2 test procedure was repeated. Phrenic activity during the last 2 min of every step change was analyzed. The CO2 sensitivity before and after sinus nerve section was determined as change in percent minute phrenic output per Torr change in arterial PCO2 from apneic threshold. Mean apneic thresholds (arterial PCO2) were not significantly different among the groups: 34.8 +/- 2.08, 32.7 +/- 2.08, and 34.7 +/- 2.25 (SE) Torr for 2- to 3-, 14- to 16-, and 21- to 22-day-old lambs, respectively. After sinus denervation, apneic thresholds were raised in all groups [39.9 +/- 2.08, 40.9 +/- 2.08, and 45.3 +/- 2.25 (SE) Torr, respectively] but were not different from each other. CO2 response slopes did not change with age before or after sinus nerve section. We conclude that carotid bodies contribute to the CO2 response during hyperoxia by affecting the apneic threshold but do not affect the steady-state CO2 sensitivity and the central chemoreceptors are functionally mature shortly after birth.

Technique for repetitive recording from fetal respiratory neurons.

We developed a new method for repetitive recording of medullary neurons in fetal sheep in situ. The technique involves chronically fixing the fetal head to the flank of the ewe by way of a Teflon plate that has a removable window. This window allows direct access of a recording electrode to the floor of the fourth ventricle of the fetus. In four of six fetuses, repetitive recordings lasting 3-4 h were possible for up to 6 days. By operating on younger fetuses and with care, this time span could be extended. This novel method should be useful in the future for extracellular and intracellular recordings of neurons in the developing fetus without disturbing the fetal state and for the study of putative neurotransmitters during development with iontophoretic techniques.

Elaboration of a conditioned reflex in a single experiment with simultaneous recording of neural activity.

In 18 of 20 experiments with click CS, and in 5 of 10 experiments with flash CS, we have elaborated a conditioned EMG response in a single session (less than 60 pairings). The US was direct electrical stimulation of the rabbit's motor cortex that evoked a movement of the forelimb. Electrical stimulation of the lateral hypothalamus was used as reinforcement. Parameters for this reinforcement were chosen to evoke a feeding reaction or self-stimulation in the freely behaving animal. The elaborated EMG response satisfied most of the required characteristics of a conditioned reflex. These included spontaneous recovery after extinction, savings of long duration, specificity to stimulus pairing, and weak efferent and afferent generalization. In most experiments with click CS, the elaborated phasic response had an amplitude of 0.1-6 mV with a latency of 12-16 msec. In form and latency the conditioned response was similar to the unconditioned startle reaction of the same animal under chloralose anesthesia, or to its unanesthetized response to a loud sound. However, the conditioned response differed from the startle reaction in that it was localized. Extracellular recordings of 2-5 neurons were simultaneously made from sensory motor cortex near the point at which the US was applied. In 7 experiments 17 neurons were followed through the entire cycle of elaboration and extinction of the conditioned response. Seven neurons showed a statistically significant (P less than 0.05) increase of the response to CS during conditioning. Latencies were 20-140 msec. Interactions between neurons were studied by computing cross-correlograms and joint PST scatter diagrams. These measures were less informative than we had hoped because of the low level of spontaneous and evoked activity, and because of the small numbers of stimulus presentations that were needed for elaboration and extinction of the conditioned reflex. In isolated presentations of the CS after elaboration of the CR, we sometimes observed neural responses to click with a latency less than 6 msec. We propose that at least one of the pathways involved in the localized conditioned startle reflex reported here goes through the sensory motor cortex.

Changes in unit postsynaptic responses at sensorimotor cortex with conditioning in rabbits.

A localized, conditioned startle reaction (LCSR) to a click (as conditional stimulus) could be established and recorded electromyographically in rabbits if complex unconditional stimulus was employed. The complex consisted of localized, electrical stimulation applied concurrently to sensorimotor cortex and hypothalamus. The latency of this LCSR was only 12-14 ms. Postsynaptic responses were recorded intracellularly or quasi-intracellularly within the sensorimotor cortex near the placement of the electrodes for the cortical stimulation. The latency distributions of the averaged postsynaptic responses were compared for the naive, conditioned and extinguished dates. Latencies in naive animals were similar to those in the extinguished state, but were significantly (P less than 0.011) different from the Latencies of click-responses in the conditioned state. An increase in the number of responses with 8-17 ms latencies was found after conditioning. Of 30 neurons recorded in conditioned animals six responded with an extremely short latency of 4 to 7 ms. Similar latencies were found in response to much more intense clicks capable of evoking an unconditioned startle reaction in naive animals. Changes in postsynaptic potentials thus occur at the cortex independent of proprioceptive feedback from the conditioned movement. The short latency neuronal responses suggest that a pathway for the LCSR may pass through the sensorimotor cortex. The appearance of responses at less than 7 ms to a previously neutral click in conditioned animals supports the idea that an increase in synaptic effectiveness underlies the neuronal mechanism of conditioning.

Maternal alcohol ingestion and the incidence of respiratory distress syndrome.

The risk of respiratory distress syndrome in infants born to mothers with varying quantities of alcohol intake during pregnancy was assessed. In infants less than 37 weeks' gestation, there was a decreasing incidence of respiratory distress syndrome with increasing maternal alcohol consumption (p less than 0.02). In addition, in infants less than 37 weeks' gestation, maternal alcohol ingestion was associated with a decreased risk of respiratory distress syndrome even when adjusted for other factors such as smoking, gestational age, birth weight, Apgar score, and sex of the infant. It is suggested that maternal alcohol ingestion enhances the maturation of the fetal lung.

Development of the EEG between 30 and 40 weeks gestation in normal and alcohol-exposed infants.

A total of 441 newborn infants with gestational ages between 30 and 40 weeks had EEG studies between 36 and 48 hours after birth. Their mothers had either abstained from alcohol during pregnancy or had ingested alcohol in one of four categories: occasional, moderate, binge or frankly alcoholic. The power of the EEG, using linear regression analysis, was significantly higher among infants of mothers in the occasional, binge and alcoholic categories than among infants of abstainers. Developmental changes in the EEGs of binge-drinking mothers were even more striking than in those of the offspring of the alcoholic mothers. These results indicate that fetal exposure to alcohol interferes with normal maturation of the brain as early as 30 weeks gestation. Furthermore, exposure to frequent high quantities of alcohol may be even more harmful to the fetal brain than continuous chronic exposure.

Prediction of subsequent motor and mental retardation in newborn infants exposed to alcohol in utero by computerized EEG analysis.

In a prospective, blinded study 38 infants of mothers with varying quantities of alcohol ingestion during pregnancy had an EEG at 40 weeks post conceptional age. Bayley Development Tests were administered between 1.5 and 10 months of age. The total power of the EEG during REM sleep was inversely related to subsequent motor development (r = -0.51, F = 13.1, p less than 0.0008) whereas the total power of the EEG during quiet sleep was inversely related to subsequent mental development (r = 0.61, F = 24.4, p less than 0.0001). In alcohol exposed babies EEG abnormalities were present even in the absence of fetal alcohol syndrome (FAS). In 16 older children born to abstainers or alcoholic mothers similar results were obtained. Thus, the power of the EEG during REM and quiet sleep at birth appears to be a sensitive index of alcohol effects on the fetal brain and may be used to predict future motor and mental development.

Effects of maternal alcohol intake and smoking on neonatal electroencephalogram and anthropometric measurements.

Anthropometric data and computerized electroencaphalogram analysis during quiet, indeterminate, and active sleep were obtained from infants of mothers of four groups: (1) heavy drinking mothers (greater than 2 ounces of alcohol per day); (2) nondrinking mothers; (3) smoking, nondrinking mothers; (4) nonsmoking, nondrinking mothers. Infants in groups 1 and 2 were matched as closely as possible for postconceptional age, sex, race, and socioeconomic status. Infants in groups 3 and 4 were matched similarly. Infants of alcoholic mothers had a significantly lower birth weight, length, and head circumference than those from the matched control group. Infants of smoking mothers had lower birth weights and lengths than infants of nonsmoking mothers, but head circumference was identical. Hypersynchrony of the electroencephalogram was seen only in "alcoholic" infants, and power spectral density analysis revealed that the average integrated power was significantly increased in quiet, active, and indeterminate sleep. The greatest increase in electroencephalogram power (212%) was seen in active sleep, and this analysis clearly separated 15 of 17 alcohol-exposed infants from the control infants. These data suggest that alcohol has a specific toxic effect on the fetal brain that is not linked with smoking habits. The neonatal electroencephalogram is affected even in the absence of dysmorphology and thus may be the most sensitive indicator of fetal alcohol toxicity.

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.