Ian R.C. Cooke

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.

Precursor of respiratory pattern in the early gestation mammalian fetus

Recordings of respiratory muscle activity in fetal lambs from early in gestation provide insight into the organization of the central pattern generator for respiration in mammals. Evidence presented here is consistent with the recent hypothesis that production of the respiratory pattern involves two separate neural modules: one, the rhythm module, which specifies the respiratory cycle and another, the form module, which creates the characteristic shape of each burst of activity within this cycle. The rhythm module is already functional when gestation is 35% complete while the form module appears to be constructed gradually over the second half of gestation.

Development of Patterns of Activity in Diaphragm of Fetal Lamb Early in Gestation

To examine the development of respiratory motor activity early in mammalian development and its relationship to nonrespiratory activity, we recorded spontaneous electromyogram activity from chronically instrumented fetal lambs over the period from 45 to 65 days gestation (G45 to G65, term = G147). Two distinct forms of motor behavior were observed at G45 in recordings made from the costal diaphragm and longissimus dorsi muscles. The predominant behavior consisted of cycles of sustained, coincident activity in the two muscles alternating with periods of inactivity. The incidence of this type of activity declined between G45 and G65 and the cyclic nature of the discharges disappeared in most animals. The second form of motor behavior at G45 consisted of episodes of repetitive bursting activity lasting up to 20 min that were confined to the diaphragm. These bursts had a duration of 97.5 +/- 8.3 ms (mean +/- S.E.M.) and frequently occurred as doublets in which two bursts were separated by an intervening period of 100-200 ms. The mean duration of these bursts declined to 69.7 +/- 7.7 ms at G65, doublets became rare, and bursts evolved a stereotyped form by G65 that was characterized by an abrupt onset and rapid decline in discharge intensity. Repetitive bursts of this form evolve into the mature respiratory motor pattern over the second half of gestation. At G45, episodes of repetitive bursting were almost always linked with episodes of sustained discharge, while at G65 these two forms of behavior were always segregated. We conclude that the neurons responsible for generating the respiratory rhythm in the lamb are assembled into a functional rhythm generator and make appropriate connections to motor output pathways as early as G45. The generation of the respiratory rhythm at G45 appears to be triggered by episodes of widespread motor activity that occur in both respiratory and nonrespiratory muscles.

Easily-implantable electrodes for chronic recording of electromyogram activity in small fetuses

A method is described for the construction of simple, robust electrodes suitable for obtaining long-term chronic recordings of electromyogram activity from delicate muscles of small experimental animals. The electrodes are equipped with small, gold-plated barbed tips which have the form of harpoons that may be pushed directly into a muscle, where they remain without being sutured into place. These features greatly facilitate the instrumentation of muscles which are very thin or of limited accessibility. The electrodes have been used successfully to obtain chronic recordings of respiratory muscle activity from early gestation fetal lambs in utero.

Supraspinal Influence on the Development of Motor Behavior in the Fetal Lamb

To examine the involvement of supraspinal inputs in the maturation of motor activity patterns in the developing fetal lamb, we recorded spontaneous electromyographic activity from spinally innervated muscles at approximately 45, 65, and 95 days gestation (G45, G65, and G95; term = 147 days). At G45, fetal activity occurred in synchronized activity-inactivity cycles of approximately 2 min duration, with the activity phase lasting 22.2 +/- 4.8 s and the inactivity phase lasting 95.4 +/- 13.3 s (mean +/- standard error of the mean, n = 5). At G65 and G95, the organization of activity was clearly different from that at G45 in that it was no longer cyclic, nor was the discharge of different muscles synchronized. By contrast, after spinal cord transection at G62, synchronised cyclic activity occurred in muscles innervated by segmental levels below the transection, both at G65 and G95. At G65 the duration of the activity phase of the cycle was 53.5 +/- 6.0 s, while the inactivity phase lasted 171.6 +/- 22.1 s; these durations did not alter between G65 and G95. Since spinal cord transection leads to the motor behavior of the G65 fetus reverting to the cyclic pattern characteristic of the G45 fetus, we conclude that supraspinal inputs begin to modulate the output of the spinal pattern generators at some stage between G45 and G65. The observation that spinally transected fetuses generate identical behavior at G65 and G95, both in terms of its cyclic character and the duration of cycles, suggests that spinal circuits undergo little autonomous development over this period; that is, the altered behavior observed in the developing intact fetus reflects the influence of supraspinal inputs on the motor circuits of the spinal cord.

The ontogeny of GABA- and glutamate-like immunoreactivity in the embryonic Australian freshwater crayfish, Cherax destructor

The distribution and ontogeny of GABA- and glutamate-like immunoreactivity in embryos of the Australian freshwater crayfish Cherax destructor were investigated over the period from 30% development until hatching. GABA-like immunoreactive cells and fibres appeared first in the brain at 40-45% development. By 70% development, GABA-like immunoreactive cells were present in almost all ganglia, and GABA-like immunoreactive fibres were distributed extensively throughout the neuropil, commissures and connectives of the central nervous system, and were also found in peripheral nerve roots supplying the appendages and the abdominal musculature. In contrast, glutamate-like immunoreactivity did not appear in the central nervous system until 60-65% development. By the time of hatching, the distribution of glutamate-like immunoreactivity was restricted to discrete regions of neuropil and fibre staining in the thoracic and abdominal nerve cord, the abdominal musculature and the appendages. The precocious establishment of the extensive distribution of GABA-like immunoreactive neurons in the developing crayfish embryo is consistent with the possibility that these neurons play a trophic role in controlling or modulating the development of the nervous system.

Differential recruitment of inspiratory muscles in response to chemical drive

Changes in the intensity of EMG activity in the costal diaphragm, crural diaphragm and external intercostal muscle during inspiration were assessed in intact, awake lambs following abrupt transitions in the composition of the inspired gases from air to either a hypoxic/hypoxic mixture (10% O2, 90% N2), a hyperoxic/hypercapnic mixture (40% O2, 6% CO2, 54% N2) or a hypoxic/hypercapnic mixture (10% O2, 6% CO2, 84% N2). A regression method was used to compare the dynamic responses of the three muscles over the 10-min period following each transition. The relationship between the dynamic response functions of the costal and crural diaphragm was the same in each of the three experimental conditions, indicating that these separate components of the diaphragm comprise a single functional unit during breathing. The relationship between the dynamic response functions of the external intercostal muscle and the costal diaphragm varied according to the composition of the inspired gas mixture. This result suggests that the central and peripheral chemoreceptors exert differential effects on the activation of the diaphragm and the external intercostal muscles during breathing, consistent with the hypothesis that sensory information from these receptors is processed, at least in part, in parallel pathways which project separately to the phrenic and external intercostal motoneurons.

The development of descending projections from the brainstem to the spinal cord in the fetal sheep

BackgroundAlthough the fetal sheep is a favoured model for studying the ontogeny of physiological control systems, there are no descriptions of the timing of arrival of the projections of supraspinal origin that regulate somatic and visceral function. In the early development of birds and mammals, spontaneous motor activity is generated within spinal circuits, but as development proceeds, a distinct change occurs in spontaneous motor patterns that is dependent on the presence of intact, descending inputs to the spinal cord. In the fetal sheep, this change occurs at approximately 65 days gestation (G65), so we therefore hypothesised that spinally-projecting axons from the neurons responsible for transforming fetal behaviour must arrive at the spinal cord level shortly before G65. Accordingly we aimed to identify the brainstem neurons that send projections to the spinal cord in the mature sheep fetus at G140 (term = G147) with retrograde tracing, and thus to establish whether any projections from the brainstem were absent from the spinal cord at G55, an age prior to the marked change in fetal motor activity has occurred.ResultsAt G140, CTB labelled cells were found within and around nuclei in the reticular formation of the medulla and pons, within the vestibular nucleus, raphe complex, red nucleus, and the nucleus of the solitary tract. This pattern of labelling is similar to that previously reported in other species. The distribution of CTB labelled neurons in the G55 fetus was similar to that of the G140 fetus.ConclusionThe brainstem nuclei that contain neurons which project axons to the spinal cord in the fetal sheep are the same as in other mammalian species. All projections present in the mature fetus at G140 have already arrived at the spinal cord by approximately one third of the way through gestation. The demonstration that the neurons responsible for transforming fetal behaviour in early ontogeny have already reached the spinal cord by G55, an age well before the change in motor behaviour occurs, suggests that the projections do not become fully functional until well after their arrival at the spinal cord.

A map of the major nuclei of the fetal sheep brainstem.

The fetal sheep has been used to investigate a wide range of developmental and pathological processes such as the effect of severe hypoxia, asphyxia, or intrauterine infection on the brain but, until now, there has been no complete description of the normal anatomical organisation of neuronal groups to facilitate interpretation of these studies. In this paper, we describe the major nuclei of the fetal sheep brainstem based on a study of 5 fetal sheep at 140 days of gestation (G140: term is G147). Nuclei were identified with the aid of brain atlases available for other species, and from the previously published, partial descriptions available for the sheep. Fifty-five distinct nuclei were identified after Nissl (thionin) staining, and their caudal and rostral margins were defined. This paper provides an easy reference to the position of the major nuclei within the fetal sheep brainstem, and can be used as a guide for future studies examining the organisation of neuronal populations under normal and pathological conditions in this animal model.

Origin of tonic electrical activity patterns in the diaphragm of the fetal lamb

The respiratory pattern generator in fetal and postnatal life activates the phrenic nucleus and diaphragm muscle with phasic bursts of activity. In the fetus, diaphragmatic activity is also characterized by tonic activity patterns of unknown origin. We have examined whether such activity is diaphragmatic, or radiated from nearby ribcage muscles, by placing two sets of electrodes side-by-side in the costal portion of the diaphragm in five fetuses. The rationale for this approach is that if tonic activity, radiates to the diaphragm it should be recorded by both sets of electrodes and there should be no delay between the action potentials from each set of electrodes. Of 24 single tonic units identified, 15 were recorded from only one of the two sets of electrodes in the diaphragm. In the 9 tonic units recorded from both sets of electrodes, there was a time delay between the appearance of the action potentials in the two recordings (mean +/- S.E.M. 1.6 +/- 0.2 ms). This is the expected conduction delay along the muscle fibres separating the two electrodes. Since tonic diaphragmatic activity persisted in fetuses with the spinal cord transected rostral or caudal to the phrenic nucleus, we conclude that the spinal cord alone is sufficient to produce the tonic activity recorded from the fetal diaphragm but that the brain may also generate such activity.