In utero model for pharmacologically investigating spontaneous activity during early ontogeny.

We describe an in utero model in which it is possible to investigate the involvement of supraspinal and spinal neurons in the genesis of spontaneous motor activity, a feature of early fetal life. To date almost all studies of the circuits that give rise to spontaneous motor activity during early ontogeny, and the neurotransmitters involved, have been carried out with in vitro models. Limitations of in vitro models include the relatively short viability of the preparation and the need to stimulate the nervous system either pharmacologically or electrically to produce the activity to be studied, in contrast to the activity that spontaneously occurs normally in utero. Our model uses fetal sheep, chronically instrumented with electromyogram electrodes and a catheter placed either intrathecally at the spinal level or in the peritoneal cavity. Motor activity can be studied over lengthy periods of fetal life and it is possible to examine the effects of infusing agonists and antagonists of central neurotransmitters on spontaneous motor activity. The use of our new model in parallel with the pre-existing in vitro models has the potential to add substantially to our understanding of the mechanisms behind changes in spontaneous activity that occur throughout fetal life.

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

BACKGROUND: Although 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. RESULTS: At 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. CONCLUSION: The 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.

Longitudinal analysis of human immunodeficiency virus type 1 nef/long terminal repeat sequences in a cohort of long-term survivors infected from a single source.

We studied the evolution of human immunodeficiency virus type 1 (HIV-1) in a cohort of long-term survivors infected with an attenuated strain of HIV-1 acquired from a single source. Although the cohort members experienced differing clinical courses, we demonstrate similar evolution of HIV-1 nef/long-terminal repeat (LTR) sequences, characterized by progressive sequence deletions tending toward a minimal nef/LTR structure that retains only sequence elements required for viral replication. The in vivo pathogenicity of attenuated HIV-1 is therefore dictated by viral and/or host factors other than those that impose a unidirectional selection pressure on the nef/LTR region of the HIV-1 genome.