Phyllis M. Gootman

Brainstem cells of origin of the cervical vagus and cardiopulmonary nerves in the neonatal pig (Sus scrofa)

The distribution of the cells of origin of the cervical vagus and cardiopulmonary nerves has been studied in neonatal piglets (Sus scrofa) ranging in age from 1 to 60 days. Cardiopulmonary nerves were identified physiologically and anatomically prior to injection of horseradish peroxidase (HRP) into the nerves. Following injection of HRP into the cervical vagus nerve retrogradely labeled neurons were present in the dorsal motor nucleus of the vagus nerve (DMV), the nucleus of the solitary tract, the nucleus ambiguus (NA), ventrolateral to the NA and in an intermediate zone between the DMV and the NA. Two unique clusters of neurons were also retrogradely labeled after injections into the vagus nerve. One group was located lateral to the most caudal levels of the DMV and extended as far caudally as the C1 spinal segment. The second distinctive group was located ventrolateral to the nucleus ambiguus in a cell column identified as the ventrolateral nucleus ambiguus (VLNA). After injections of HRP into cardiopulmonary nerves, the majority of neurons were found in the VLNA and the distinct clusters of neurons in this cell column were particularly heavily labeled. Small numbers of cells were labeled in the DMV and NA and none were labeled in the solitary nucleus after cardiopulmonary nerve injections. There were no apparent age-related differences in the degree or distribution of retrograde labeling. The distribution of neurons in the medulla oblongata projecting into cardiopulmonary nerves in the piglet is similar to that described in other species, i.e., the nucleus ambiguus, particularly its ventrolateral cell column, is the primary site of cardiomotor neurons. In addition, in the piglet there is a morphologically distinct cluster of cells related to the heart, and possibly the lungs, which does not appear to be present in other species.

Anatomy of medullary and peripheral autonomic neurons innervating the neonatal porcine heart.

Gross and microscopic anatomical investigations were carried out in 14 piglets aged from 4 to 66 days. True Blue (7-50 microliters) and Diamidino Yellow (7-50 microliters) were injected individually into 2 different cardiac sites (the right atrial ganglionated plexus, the inferior vena cava, inferior atrial ganglionated plexus, the right atrium or the right ventricle). Gross anatomy: Globular superior cervical and nodose ganglia, elongated stellate ganglia, multiple small middle cervical ganglia and multiple small mediastinal ganglia along the course of cardiopulmonary nerves were identified. Microscopic anatomy: Neurons innervating specific cardiac regions or intrinsic cardiac ganglionated plexuses were distributed relatively evenly among stellate (primarily in their cranial poles) and middle cervical ganglia bilaterally, fewer labeled neurons being located in the superior cervical and mediastinal ganglia bilaterally. Parasympathetic efferent preganglionic neurons associated with either intrinsic cardiac ganglionated plexus studied were identified primarily throughout the ventrolateral region (the external formation) of the nucleus ambiguus bilaterally. Labeled neurons were also identified throughout the right and left nodose ganglia. Individual neurons did not project axons to different cardiac regions, as no double-labeled neurons were identified. No correlation between age and the numbers and locations of labeled neurons was apparent. Thus, porcine sympathetic efferent neurons which innervate individual cardiac regions, including intrinsic cardiac ganglionated plexuses, lie scattered primarily throughout the right and left mediastinal and middle cervical ganglia as well as the cranial poles of stellate ganglia at birth, apparently changing little during the first 2 months of age. Porcine cardiac parasympathetic efferent preganglionic neurons are located primarily in the external formation of the nucleus ambiguus bilaterally at birth. The numbers of afferent cardiac neurons distributed throughout the nodose ganglia bilaterally also change little during that time. It is concluded that most of the autonomic neurons which innervate the heart are in place at birth.

A search for medullary neurons with activity patterns similar to those in sympathetic nerves

The existence of common activation of different sympathetic outflows is implied by the presence of similar periodicities, usually cardiac and/or respiratory, in different sympathetic nerves, as reported in our own work 8,15,Is,Is, and by other investigators21,26,28,30,38, 43. In our own studies simultaneous recordings from 2 sympathetic nerves showed that they had common periodicities which were phase-locked, e.g., 10/sec, cardiac, and respiratory rhythms, which presumably arose from supraspinal sources ls,la. Therefore, in the present study we have attempted to find these driving sources in the medullary reticular formation by searching for neurons whose activity is temporally related to sympathetic discharge. Experiments were performed on decerebrate or urethane anesthetized (1.0-1.25 g/kg, i.v.) cats (2.5-4.3 kg) with neuromuscular blockade (gallamine or decamethonium), bilateral vagotomy, bilateral pneumothorax and artificial ventilation. For further details, see earlier papersS,16,17. Recordings were made on magnetic tape and on an Offner inkwriter of: (a) efferent left greater splanchnic (SPL) nerve activity; (b) efferent left cervical sympathetic (CS) nerve activity (both activities recorded monophasically with bipolar electrodes and using a 0.2-1000 Hz bandpass); (c) efferent phrenic (PHR) discharge (bandpass 80-I0,000 Hz); (d) pulses derived from the PHR neurogram to mark the start of the inspiratory (I) and expiratory (E) phasesS,15; (e) electrocardiogram (EKG); (f) standard pulses derived from the R-wave of the EKG; (g) thoracic aortic pressure; (h) activity of brain stem neurons (extracellularly recorded by insulated tungsten microelectrodes, with tip diameter 1.0/zm and resistances 2-5 Mf~); (i) standard pulses derived from the spikes by means of an amplitude discriminator. To verify that a recording was from an isolated unit, the microelectrode signal was passed through a digital delay circuit and displayed in sweeps triggered by the derived pulses, so that the total waveform could be seen. In some experiments monopolar stimulation (single pulses, 0.1 msec duration, 0.2-0.5 mA) was applied through the microelectrode. Data analysis. The time relations of SPL, CS, PHR, and unit activity were

Cardiac responses elicited by stimulation of loci within stellate ganglia of developing swine

Stimulation with bipolar electrodes of specific loci in stellate ganglia elicited in anesthetized piglets, 1-4 weeks of age, alterations in cardiac function and aortic pressure. Responses were also elicited by chemical stimulation in specific loci of these ganglia. The probability of eliciting a cardiovascular response by stimulating loci in a stellate ganglion increased with increasing postnatal age. For instance, no responses were elicited when loci in the left stellate ganglia of 1-week-old piglets were stimulated. Significant heart rate responses were obtained only when loci in right stellate ganglia were stimulated. The number of ganglionic loci from which cardiovascular responses were obtained increased with increasing postnatal age. It is concluded that the capacity of stellate ganglion neurons to modulate the cardiovascular system matures during the first four weeks of life, heart rate being modulated primarily by neurons in the right stellate ganglion and inotropism by neurons in both stellate ganglia.

Presumptive adrenergic neurons containing phenylethanolamine N-methyltransferase immunoreactivity in the medulla oblongata of neonatal swine.

Given the importance of the swine (Sus scrofa) as an animal model for human development, physiology and disease, neurons containing the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT), were mapped in the medulla oblongata of neonatal swine as a first step in identifying their roles in central autonomic control. Neurons were labeled immunocytochemically by using an antiserum to PNMT raised in rabbits against trypsin-treated enzyme purified from the bovine adrenal gland. The general regional organization of neurons expressing PNMT (-like) immunoreactivity (ir) in the neonatal swine was similar to data obtained in other species and, in some aspects, more closely resembled the pattern observed in the primate brain. Immunolabeled cells appeared to be more abundant and caudally more extensive than observed in other adult animals. PNMT-immunoreactive (ir) neuronal somata, however, were largely confined to the reticular formation in the ventrolateral quadrant and the nucleus tractus solitarii (NTS) and more restricted in distribution than those expressing tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (D beta H)-ir on serial transverse sections. A close correspondence was observed between the distributions of TH- and PNMT-ir neurons and processes throughout the C1 and C2 areas. However, in the C1 and C3 regions TH-ir neurons outnumbered those containing D beta H and PNMT-ir. In contrast, cell groups enriched in PNMT-ir neurons and processes were characterized by relatively weak D beta H-ir. In the ventrolateral medulla (VLM), PNMT-ir cell bodies were concentrated rostrally and extended from the caudal pole of the facial nucleus to a level posterior to the calamus scriptorius. The rostral VLM was characterized by an admixture of bipolar and multipolar primarily medium-diameter immunostained neurons. A prominent cell column (condensation) organized ventromedially to the nucleus ambiguus pars compactus (NAc). A loosely organized cluster bordered the lateral aspect of the special visceral efferent column; another smaller aggregate was located in the ventromedial reticular formation adjacent to the inferior olive. At middle medullary levels, PNMT-ir neurons formed two distinct subgroups (dorsal and ventral) interrupted by a band of precerebellar relay neurons that extended between the medial and lateral limbs of the lateral reticular nucleus of Walberg. At obex, the dorsal cell group formed a diagonal array and assumed a position dorsal and dorsolateral to the medial limb of LRN. This group was distinguished by bipolar neurons with axes of orientation directed perpendicularly to the majority of neurons in the rostal VLM or those lying near the caudal ventromedullary surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Age-related changes in power spectra of efferent phrenic activity in the piglet

Power spectral analysis of phrenic nerve discharge in neonatal swine revealed the presence of both high-frequency oscillations (HFO) (95-150 Hz) and medium-frequency oscillations (MFO) (15-35 Hz). The HFO was shown to be age-related; the MFO was not. The data indicated that at least one manifestation of maturation of the respiratory rhythm generator is the increase with age of the frequency of the HFO.

CO2-induced expression of c-fos in the nucleus of the solitary tract and the area postrema of developing swine

This investigation was performed to determine whether hypercapnic exposure elicited expression of the c-fos protooncogene product, FOS, in nucleus of the solitary tract (NTS) and area postrema (AP) neurons of developing swine. Mean arterial blood pressure (MAP) and heart rate (HR) were also monitored to evaluate whether numbers of neurons containing FOS were related to changes of MAP and HR. In each experiment, two litter-matched piglets were prepared simultaneously, i.e., Saffan anesthesia, paralysis, and artificial ventilation (100% O(2)). One animal was exposed to hypercapnia (1 h of 10% CO(2), balance oxygen), while the other continued to breathe 100% O(2). Animals were studied at three different ages: 5-8 days, 13-15 days, and 26-34 days old. In the NTS, FOS expression was prominent in regions corresponding to the general visceral afferent subdivision; the AP showed no such topographic distribution. The number of NTS and AP neurons with FOS in hypercapnic-exposed animals was significantly greater than those of unexposed animals. However, an age-related increase of FOS was observed only for NTS neurons, with the greatest number observed in 13- to 15-day-old animals. Increases of MAP, not HR, were noted during the early part of hypercapnia in the 5- to 8-day-old group; older animals exhibited no change of MAP. Our findings demonstrated that prolonged hypercapnic stimulation elicited FOS expression in AP and NTS neurons of developing animals, and that such expression was non-uniform, depending upon the region studied.

Pulmonary Afferent Influences on Respiratory Modulation of Sympathetic Discharge

Experiments were performed on decerebrate or urethane-anesthetized, gallamine-paralyzed cats with pneumothorax and intact vagi. Efferent splanchnic and cervical sympathetic nerve discharges were recorded monophasically (0.2–1000 HZ). Phrenic nerve discharge served as an indicator of respiratory center output. Lung inflation was applied co-incidentally with phrenic discharge during control cycles by means of a cycle-triggered pump. Changes in the timing of cycle-triggered pump inflations were used to evaluate effects of pulmonary afferent activity from lung stretch receptors on central respiratory modulation of sympathetic discharge. When inflation was not applied for one inspiratory phase, the Breuer-Hering inspiratory-inhibitory reflex did not occur: inspiratory prolongation with no change in slope of the integral of phrenic activity, as measured with an average-response computer. In contrast, this test produced an increase in slope of the integrated sympathetic discharge. This indicates that inflations during the control inspiratory phases inhibited sympathetic discharge. The striking difference in the slope of phrenic vs sympathetic activities implies that this inhibition of sympathetic activity was acting via circuits different from, but related to, those of the Breuer-Hering reflex. Lung inflation also inhibited sympathetic discharge during the expiratory phase, since inflations applied during the expiratory phase reduced sympathetic discharge, concomitantly with lengthening of the expiratory phase (Breuer-Hering expiratory-facilitatory reflex). The latency for sympathetic inhibition from the onset of inflation was of the order of 100–200 ms both for inspiratory and expiratory inflations. Vagotomy abolished these effects and resulted in an increased respiratory modulation of sympathetic discharge. These results indicate that pulmonary afferent activity exerts an important influence on the central respiratory modulation of sympathetic discharge.

Effects of elevated plasma magnesium concentration on cerebrospinal fluid levels of magnesium in neonatal swine.

Abstract To determine whether magnesium (Mg) can cross the blood brain barrier in developing swine, simultaneous measurements of [Mg] in plasma and cerebrospinal fluid (CSF) were made during experimental elevation of plasma [Mg] in 12 swine of differing postnatal age. All were anesthetized with Saffan and maintained at normal arterial blood gas composition. Aortic pressure and heart rate were monitored. Plasma and CSF samples, drawn at the beginning and end of a 60-min intravenous infusion of MgCl2 in all animals and every 10 min during the infusion in three, were analyzed for [Mg] and osmolality. CSF [Mg] increased in all animals as plasma [Mg] increased. There were no changes in CSF osmolality. The differences between plasma and CSF [Mg] was smallest in the youngest animals. These results indicate that Mg crosses the blood brain barrier in neonatal swine and suggest that the blood brain barrier is still maturing within the first postnatal month.

Neuronal activity of the stellate ganglia in neonatal swine

In anesthetized, paralyzed, and artificially ventilated neonatal pigs, neuronal activity was recorded extracellularly from the intact right stellate ganglion. Of 301 investigated neurons, only a few had spontaneous activity: 27 neurons generated activity either during lung inflation or deflation, and 1 neuron generated activity related to the cardiac cycle. The remaining 273 neurons were activated by stroking of body hair or by light pressure applied to various somatic sites. These data suggest that much of the spontaneous activity in the stellate ganglia appear later during development.