Cell Death During Development of the Nervous System

Neuron and synapse numbers are important assays in neuroscience. These numbers are estimated by one of four methods: 1) profile counts, 2) assumption-based methods, 3) serial reconstructions, and 4) stereological methods. The criteria for these methods are diverse. This creates a disparity in that some reviewers accept estimates from any of these methods, while others accept only specific methods. An equally important issue is the diversity of sampling strategies, since unbiased estimates of neuronal or synaptic numbers are contingent upon both counting and sampling techniques. The purpose of this commentary is to institute a dialog that will lead to a better understanding of the strengths and weaknesses of the above methods, and to propose guidelines that should lead to more uniform and thus fairer judging of the studies that provide estimates of neuron or synapse numbers. In addition, adoption of more uniform standards for obtaining unbiased numerical estimates should result in the generation of an unbiased database that will be of considerable use in future studies.

Methods for determining numbers of cells and synapses: A case for more uniform standards of review

We have examined the hypothesis that neuronal programmed cell death requires a genetic program; we used a model wherein rat sympathetic neurons maintained in vitro are deprived of NGF and subsequently undergo apoptosis. To evaluate gene expression potentially necessary for this process, we used a PCR-based technique and in situ hybridization; patterns of general gene repression and selective gene induction were identified in NGF-deprived neurons. A temporal cascade of induced genes included "immediate early genes," which were remarkable in that their induction occurred hours after the initial stimulus of NGF removal and the synthesis of some required ongoing protein synthesis. The cascade also included the cell cycle gene c-myb and the genes encoding the extracellular matrix proteases transin and collagenase. Concurrent in situ hybridization and nuclear staining revealed that while c-jun was induced in most neurons, c-fos induction was restricted to neurons undergoing chromatin condensation, a hallmark of apoptosis. To evaluate the functional role of the proteins encoded by these genes, neutralizing antibodies were injected into neurons. Antibodies specific for either c-Jun or the Fos family (c-Fos, Fos B, Fra-1, and Fra-2) protected NGF-deprived neurons from apoptosis, whereas antibodies specific for Jun B, Jun D, or three nonimmune antibody preparations had no protective effect. Because these induced genes encode proteins ranging from a transcription factor necessary for death to proteases likely involved in tissue remodeling concurrent with death, these data may outline a genetic program responsible for neuronal programmed cell death.

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Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis.

https://www.cell.com/neuron/pdf/0896-6273(95)90331-3.pdf

A c-jun dominant negative mutant protects sympathetic neurons against programmed cell death

Organizational and activational effects of sex steroids on brain and behavior: A reanalysis

Published 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.

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

Counts 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.

Linda L. Wright

Papers

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

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

Neonatal testosterone treatment increases neuron and synapse numbers in male rat superior cervical ganglion

Effects of 17-β-estradiol on developing superior cervical ganglion neurons and synapses

Neurotransmitter synthesis, storage, and turnover in neonatally deafferented sympathetic neurons.