Timothy J. Cunningham

Naturally occurring neuron death and its regulation by developing neural pathways.

Publisher Summary This chapter describes the naturally occurring neuron death and its regulation by developing neural pathways. The chapter describes degenerating neurons and shows how the morphological expression of cell death is closely related to a neurons state of maturity. The timing of natural cell death is also considered to define where natural neuron death fits into other aspects of neural development and neuron differentiation. A large proportion of neurons that are destined to die naturally have a capacity to differentiate normally. Experimental studies show the dependence that a neuron has on the cell population with which it ultimately connects. Removal of targets for certain populations of neurons accentuates the normal attrition of neurons during development. The chapter also discusses the possible functions of cell death to provide a comprehensive explanation for this phenomenon.

Neuron numbers in the superior cervical sympathetic ganglion of the rat: a critical comparison of methods for cell counting

SummaryPublished values for the number of neurons in the superior cervical ganglion of the adult rat range from 13 000 to 45 000. These studies have employed different methods for determining what unit to count (cell body, nucleus, nucleolus), how many sections to count, and how to correct the raw counts for split particles and for profiles that are too small to resolve. The purpose of this study was to examine the extent to which these parameters may influence the calculated value for the total number of neurons, using computer simulations of neuron populations. These simulations permitted us to determine the effects on neuron number of varying the diameter of the neuronal nucleus, the size of the smallest resolvable profile, and the thickness of the section. The data from the simulations were used to test the validity of several methods that are in common use for correction of neuron counts. Our results indicate that most of the methods that are in routine use are unsatisfactory. We propose the use of either one of two methods that consistently result in highly accurate estimates of neuron numbers. These are: (1) a modification of the method proposed by Hendry (1976), using computer analysis; or (2) a modification of the method proposed by Abercrombie (1946), which does not require the use of a computer.

Naturally occurring neuron death in the ganglion cell layer of the neonatal rat: morphology and evidence for regional correspondence with neuron death in superior colliculus.

Abstract Light and electron microscopic examination of the inner layers of the normal infant rat retina reveal naturally degenerating cell bodies in the ganglion cell layer, degenerating axons in the optic fiber layer, and degenerating processes in the inner plexiform layer. The degenerating ganglion cell bodies are characterized by condensed nuclear chromatin, an abundance of free monosomes, and few cisternae of rough endoplasmic reticulum. The cells correspond to one type of degenerating neuron described in normal bird embryos but are surprisingly quite different from the morphology of the majority of naturally degenerating neurons in the infant rat superior colliculus. The reason for this difference appears related to the relative immaturity of the ganglion cells when they die. By inspection of the present material and from previous results14 it is clear that the timing of cell death in the ganglion cell layer and colliculus overlaps considerably. Furthermore, counts of degenerating profiles in 6-and 7-day-old rats (the days when cell death appears maximal in both structures) show that caudal superior colliculus and nasal ganglion cell layer, which are normally connected, have significantly more degenerating profiles than rostral colliculus and temporal ganglion cell layer, which are also connected. It may be that these corresponding patterns of cell death reflect a reciprocal control of neuron survival in the two structures. In the course of our study of degenerating cells we also find evidence for two populations of normal neurons in the developing ganglion cell layer. One is a large pale cell which is relatively rich in intracytoplasmic organelles and the other is a small, more densely staining cell which has relatively sparce organelles and coarse nuclear chromatin (at least at the earlier postnatal ages). The possibility exists that this second cell type represents an interneuron located in the ganglion cell layer.

Developmental neuron death in the rat superior cervical sympathetic ganglion: cell counts and ultrastructure

SummaryCounts of neurons of the rat superior cervical ganglion (SCG) were made at two days before birth and at several postnatal ages. There is a significant decline in the number of apparently normal neurons over the first postnatal week, with the number falling from 39 500 at 3 days to 26 500 at 7 days. Cell numbers then remained constant up to day 60 when the number of neurons was 27 500. The incidence of degenerating neurons, identified by light and electron microscopy, was correlated temporally with the loss of normal neurons. The early manifestations of the neuron degeneration were chromatin clumping and the presence of free monoribosomes. Later stages were characterized by increased chromatin clumping, dense aggregations of monoribosomes, numerous intracytoplasmic vacuoles, and only short segments of rough endoplasmic reticulum. The ultrastructure of the majority of these dying neurons is similar to the ‘nuclear’ types of degeneration described by Pilar & Landmesser (1976) and Chu-Wang & Oppenheim (1978). Based on the presence of degenerating neurons coincident with the reduction in neuron numbers, we conclude that neuron death is an important aspect of early postnatal development in the rat SCG.

Naturally occurring neuron death in the optic layers of superior colliculus of the postnatal rat.

SummaryApplication of light and electron microscopic techniques to the superior colliculus of the normal rat shows that a number of neurons die within the first week after birth. Cells in the earliest recognizable stages of degeneration are characterized by an overall increase in electron density and dilation of the intracellular cisternae, although there are only minimal changes in the chromatin pattern of the nucleus. Synapses are found on these cells. A second type of degenerating cell, with more striking changes in the nucleus, also appears in the tissue but very infrequently. In later stages of degeneration, cells are reduced to a condensed chromatin mass surrounded by disrupted fragments of the rest of the cell. This ‘cellular debris’ is found within glial cytoplasm. The majority of these debris profiles appear in the first postnatal week and are usually most abundant in the caudal third compared to either the rostral or middle thirds of the colliculus. The results suggest that the mechanisms controlling neuron number in the superior colliculus are operative in the postnatal rat but argue against a simple relationship between the survival of a particular neuron and the total number of optic connections that neuron has received.

Cortical transplants reveal CNS trophic interactions in situ

Five days after transplanting fetal rat posterior cortex into newborn host rats with posterior cortex lesions, the hosts dorsal lateral geniculate nucleus (dLGN) contains 2-5 times as many neurons as the dLGN of lesion-only controls. This effect is temporary and restricted to neurons created later in dLGN neurogenesis. Similar transplants of cerebellar plate are ineffective. These findings suggest that intracranial transplants of CNS tissue can be a source of specific trophic support to particular host neurons.

Identification of the human cDNA for new survival/evasion peptide (DSEP): Studies in vitro and in vivo of overexpression by neural cells

We identified the human cDNA encoding a peptide that has been partially purified from the secretions of oxidatively stressed neural cell lines, murine adenocarcinoma cells, and group Abeta-hemolytic steptococci. We then genetically modified mouse and human neural cells to overexpress this peptide and found these modified cells to be remarkably hearty, surviving under conditions of severe oxidative stress, in xenocultures when exposed to activated macrophages, and as xenografts in the brain of rats that were not immunosuppressed. The peptide is called DSEP (dee-sep) for diffusible survival evasion peptide. Part of the survival advantage of DSEP overexpressors may be due to their attenuated response to all-trans-retinoic acid, which regulates differentiation and apoptosis of several cell types including neural and immune cells.

Diffusible proteins prolong survival of dorsal lateral geniculate neurons following occipital cortex lesions in newborn rats

Removal of the occipital cortex in newborn rats results in the rapid and nearly complete degeneration of the dorsal lateral geniculate nucleus (dLGN) in 5 days. In previous studies we have shown that transplants of embryonic posterior cortex neurons, which are allowed to develop in culture for 5 days prior to transplantation into the site of the lesion, prolong the survival of a particular population of host dLGN neurons for an additional week. In this study we tested the possibility that the transplant cells synthesize diffusible proteins which are responsible for this neurotrophic effect. Culture medium conditioned by explants of embryonic occipital cortex and diencephalon was concentrated by vacuum dialysis or ultrafiltration through membranes with at least a 10-kDa cut-off. This concentrated medium was loaded into polyacrylamide or sodium alginate gels which were then implanted into the cavity of the lesion. Five days after implantation, the alginate-conditioned-medium implants result in a 3-fold increase in dLGN survival compared to unconditioned medium controls, while a two-fold increase in survival of the nucleus is found with the polyacrylamide-conditioned-medium implants. Proteolysis of the conditioned medium eliminates all neurotrophic activity. The results suggest that the death of dLGN neurons following the cortical lesion is due to the loss of diffusible proteinaceous neurotrophic factors--factors that may operate during normal in vivo development of the geniculocortical pathway.

Microglial invasion and activation in response to naturally occurring neuronal degeneration in the ganglion cell layer of the postnatal cat retina

Retinae of kittens between postnatal (P) days 2 and 10 were examined for the presence of degenerating neuronal profiles, normal nucleoli and microglia. Comparison of the numbers of degenerating profiles with numbers of axons lost from the optic nerve suggest that the majority of these profiles result from the degeneration of retinal ganglion cells. Analysis of local densities of the different profiles revealed different rates of cell loss, occurring at different times in central and peripheral retina. The period of rapid cell loss occurred between P2 and P3 in central retina compared to between P8 and P10 in peripheral retina. At both locations, these periods of rapid cell loss were accompanied by a decrease in the ratio of microglia to dying cells even though the absolute densities of microglia increased. However, calculation of the clearance times of cellular debris indicate that the speed of removal of degeneration products is greater during rapid cell loss, which suggests that cellular degeneration serves to activate the phagocytic process.

Calreticulin Binding and Other Biological Activities of Survival Peptide Y-P30 Including Effects of Systemic Treatment of Rats

Neuron survival-promoting peptide Y-P30, purified from oxidatively stressed neural cell lines, inhibits the appearance of microglia and rescues neurons 1 week after direct application to lesions of the rat cerebral cortex (7). Y-P30 affinity matrices treated with solubilized membranes from a variety of cell lines including human neuroblastoma SY5Y, mouse hippocampal cells HN 33.1, and human promonocytes HL-60, as well as with cerebral cortex tissue from both humans and rats, showed highly specific binding to calreticulin, a ubiquitous calcium binding protein that may be critical for integrin function. Treatment of cultures with 0.1 nM Y-P30 stabilized all these cell types whether differentiated or not, while 1 microM peptide also inhibited the morphological differentiation of the HL-60 cells into macrophages. Western analysis of the medium of SY5Y cell cultures suggested Y-P30-stimulated release of calreticulin, a result consistent with its other biological activities. Likewise, single dose systemic application of Y-P30 in unoperated rats and in rats with cerebral cortex lesions produced significant reductions in cerebral cortex membrane-associated calreticulin. Both direct and intravenous treatment with peptide also reduced cortical neuron atrophy 4 days after these lesions but only direct application consistently inhibited the appearance of ED-1(+) monocyte derivatives. We suggest that in vitro and in vivo mechanisms of Y-P30 effects are similar and involve the targeting of calreticulin. The results also suggest that some of these activities are apparent in the cerebral cortex after systemic application of this peptide.