Jane L. Lubischer

A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function

Understanding the cell–cell interactions that control CNS development and function has long been limited by the lack of methods to cleanly separate neural cell types. Here we describe methods for the prospective isolation and purification of astrocytes, neurons, and oligodendrocytes from developing and mature mouse forebrain. We used FACS (fluorescent-activated cell sorting) to isolate astrocytes from transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of an S100β promoter. Using Affymetrix GeneChip Arrays, we then created a transcriptome database of the expression levels of >20,000 genes by gene profiling these three main CNS neural cell types at various postnatal ages between postnatal day 1 (P1) and P30. This database provides a detailed global characterization and comparison of the genes expressed by acutely isolated astrocytes, neurons, and oligodendrocytes. We found that Aldh1L1 is a highly specific antigenic marker for astrocytes with a substantially broader pattern of astrocyte expression than the traditional astrocyte marker GFAP. Astrocytes were enriched in specific metabolic and lipid synthetic pathways, as well as the draper/Megf10 and Mertk/integrin αvβ5 phagocytic pathways suggesting that astrocytes are professional phagocytes. Our findings call into question the concept of a “glial” cell class as the gene profiles of astrocytes and oligodendrocytes are as dissimilar to each other as they are to neurons. This transcriptome database of acutely isolated purified astrocytes, neurons, and oligodendrocytes provides a resource to the neuroscience community by providing improved cell-type-specific markers and for better understanding of neural development, function, and disease.

Fluorescent Proteins Expressed in Mouse Transgenic Lines Mark Subsets of Glia, Neurons, Macrophages, and Dendritic Cells for Vital Examination

To enable vital observation of glia at the neuromuscular junction, transgenic mice were generated that express proteins of the green fluorescent protein family under control of transcriptional regulatory sequences of the human S100B gene. Terminal Schwann cells were imaged repetitively in living animals of one of the transgenic lines to show that, except for extension and retraction of short processes, the glial coverings of the adult neuromuscular synapse are stable. In other lines, subsets of Schwann cells were labeled. The distribution of label suggests that Schwann cells at individual synapses are clonally related, a finding with implications for how these cells might be sorted during postnatal development. Other labeling patterns, some present in unique lines, included astrocytes, microglia, and subsets of cerebellar Bergmann glia, spinal motor neurons, macrophages, and dendritic cells. We show that lines with labeled macrophages can be used to follow the accumulation of these cells at sites of injury.

EVIDENCE FOR TARGET REGULATION OF THE DEVELOPMENT OF ANDROGEN SENSITIVITY IN RAT SPINAL MOTONEURONS

Specific neuronal circuits within the vertebrate nervous system express high levels of steroid receptors and are sensitive to the effects of steroid hormones. The mechanisms by which these neuronal circuits develop their unique steroid sensitivity are unknown. One intriguing hypothesis is that retrograde influences during early postnatal life play a role in determining which central nervous system (CNS) neurons become sensitive to steroids. We now present evidence that during a critical period in early postnatal development, axonal injury disrupts the normal development of steroid sensitivity. The spinal nucleus of the bulbocavernosus (SNB) is a neuromuscular system that is highly androgen-sensitive at the level of both the motoneurons and their target muscles. Testosterone levels regulate the size of SNB motoneurons and their muscles in adult rats. Cutting the axons of SNB motoneurons on postnatal day 14 (P14) caused permanent decreases in SNB motoneuronal soma size, as well as in SNB target muscle weight. Interestingly, SNB motoneurons that survived axotomy on P14 failed to develop their normal ability to respond to testosterone in adulthood. That is, they did not respond to changes in testosterone levels with changes in soma size. The same effect was not seen after axotomy 1 week later in development, suggesting a critical period for this effect. Thus, separation from the target muscles during an early critical period in development blocked the differentiation of androgen sensitivity by SNB motoneurons, consistent with a role for the target in the normal development of steroid sensitivity by CNS neurons.

Axotomy transiently down-regulates androgen receptors in motoneurons of the spinal nucleus of the bulbocavernosus

Testosterone is an important trophic factor for motoneurons in the spinal nucleus of the bulbocavernosus (SNB), and SNB motoneurons are more responsive to testosterone than are other motoneurons. Axonal injury during early postnatal life prevents the normal development of steroid-sensitivity by adult SNB motoneurons. Axonal injury also causes changes in the expression by motoneurons of a wide range of proteins, including the up-regulation of trophic factor receptors. We have used a polyclonal antibody (PG-21; G.S. Prins) to study the expression of androgen receptors in SNB motoneurons after axonal injury. PG-21 labeled motoneuronal nuclei in the lower lumbar spinal cord of rats in a pattern that matched autoradiographic reports of androgen accumulation in this region of the nervous system. A population of numerous, small cells located dorsal to the central canal also showed evidence of androgen receptor expression. Cutting the axons of SNB motoneurons in adulthood or in development caused a decrease in androgen receptor immunoreactivity in SNB motoneurons. This is the first report that a trophic factor receptor in motoneurons is down-regulated after axonal injury, and is interesting in light of reports that testosterone treatment can facilitate motoneuronal regeneration after nerve cut. Androgen receptor levels subsequently returned to normal, regardless of the age at axotomy, providing no evidence for a lasting effect of developmental axotomy on androgen receptor levels in SNB motoneurons. Thus, axotomy-induced down-regulation of androgen receptors does not underlie the inability of SNB motoneurons to respond to androgen treatment several months after pudendal nerve cut in development.

Effect of N‐acetylaspartylglutamate (NAAG) on non‐quantal and spontaneous quantal release of acetylcholine at the neuromuscular synapse of rat

N‐Acetylaspartylglutamate (NAAG), known to be present in rat motor neurons, may participate in neuronal modulation of non‐quantal secretion of acetylcholine (ACh) from motor nerve terminals. Non‐quantal release of ACh was estimated by the amplitude of the endplate membrane hyperpolarization (H‐effect) caused by inhibition of nicotinic receptors by (+)‐tubocurarine and acetylcholinesterase by armin (diethoxy‐p‐nitrophenyl phosphate). Application of exogenous NAAG decreased the H‐effect in a dose‐dependent manner. The reduction of the H‐effect by NAAG was completely removed when N‐acetyl‐β‐aspartylglutamate (βNAAG) or 2‐(phosphonomethyl)‐pentanedioic acid (2‐PMPA) was used to inhibit glutamate carboxypeptidase II (GCP II), a presynaptic Schwann cell membrane‐associated ectoenzyme that hydrolyzes NAAG to glutamate and N‐acetylaspartate. Bath application of glutamate decreased the H‐effect similarly to the action of NAAG but N‐acetylaspartate was without effect. Inhibition of NMDA receptors by dl‐2‐amino‐5‐phosphopentanoic acid, (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzocyclohepten‐5,10‐imine (MK801), and 7‐chlorokynurenic acid or inhibition of muscle nitric oxide synthase (NO synthase) by NG‐nitro‐l‐arginine methyl ester and 3‐bromo‐7‐nitroindazole completely prevented the decrease of the H‐effect by NAAG. These results suggest that glutamate, produced by enzymatic hydrolysis of bath‐applied NAAG, can modulate non‐quantal secretion of ACh from the presynaptic terminal of the neuromuscular synapse via activation of postsynaptic NMDA receptors and synthesis of nitric oxide (NO) in muscle fibers. NAAG also increased the frequency of miniature endplate potentials (mEPPs) generated by spontaneous quantal secretion of ACh, whereas the mean amplitude and time constants for rise time and for decay of mEPPs did not change.