Expression of nestin by neural cells in the adult rat and human brain

Abstract

Neurons and glial cells in the developing brain arise from neural progenitor cells (NPCs). Nestin, an intermediate filament protein, is thought to be expressed exclusively by NPCs in the normal brain, and is replaced by the expression of proteins specific for neurons or glia in differentiated cells. Nestin expressing NPCs are found in the adult brain in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus. While significant attention has been paid to studying NPCs in the SVZ and SGZ in the adult brain, relatively little attention has been paid to determining whether nestin-expressing neural cells (NECs) exist outside of the SVZ and SGZ. We therefore stained sections immunocytochemically from the adult rat and human brain for NECs, observed four distinct classes of these cells, and present here the first comprehensive report on these cells. Class I cells are among the smallest neural cells in the brain and are widely distributed. Class II cells are located in the walls of the aqueduct and third ventricle. Class IV cells are found throughout the forebrain and typically reside immediately adjacent to a neuron. Class III cells are observed only in the basal forebrain and closely related areas such as the hippocampus and corpus striatum. Class III cells resemble neurons structurally and co-express markers associated exclusively with neurons. Cell proliferation experiments demonstrate that Class III cells are not recently born. Instead, these cells appear to be mature neurons in the adult brain that express nestin. Neurons that express nestin are not supposed to exist in the brain at any stage of development. That these unique neurons are found only in brain regions involved in higher order cognitive function suggests that they may be remodeling their cytoskeleton in supporting the neural plasticity required for these functions.

Figure 1. Class I, II and IV cells.

A typical Class I cell, immunostained chromogenically for nestin, with a very small cell soma and few sparse processes (A). Class II cells, stained for nestin chromogenically and counter-stained with cresyl violet, clustered in the wall of the third ventricle (B). Class II cells stained for nestin chromogenically in the medial wall of the lateral ventricle near its merger with the third ventricle (C). The lateral wall of the lateral ventricle directly opposite the medial wall shown in C. Note the absence of Class II cells in the lateral wall (D). Class IV cell stained chromogenically. Note the small number of very fine processes (E). Immunofluorescence staining of a Class IV cell (green) and three NeuN⁺ neurons (red) (F). The merged image in the right panel shows that the Class IV cell abuts one of the neurons. Scale bar: 5 µm (A) and (E), 10 µm (B), 20 µm (C) and (D), 15 µm (F).

Figure 2. Cell soma size and density of Class I cells and CD11b⁺ microglia.

The number of Class I nestin⁺ cells is increased twentyfold in the immediate vicinity of a cortical lesion (A). Histograms showing the density of nestin⁺ Class I cells and CD11b⁺ microglia in the neocortex of a normal rat (B). The density of microglial cells is more than fifteen times greater than that of Class I cells. Histograms showing the mean cell soma cross sectional area for nestin⁺ Class I cells and CD11b⁺ microglia (C). The mean cell soma size of microglial cells is 1.5 times that of Class I cells. The difference in mean cell soma size between Class I cells and microglia is significant (p <.001, two tailed t-test). Scale bar: 20 µm.

Figure 3. Class IV cells do not express typical neural cell markers.

Class IV cells, which express nestin (green), do not express the neural cell markers (red): NeuN (A), βIII-tubulin (B), doublecortin (C), GST-π (D), SOX-2 (E), or NG2 (F). Insets show images of cells expressing the specific marker collected using the same confocal acquisition settings. Scale bar: 15 µm (30 µm for the insets).

Figure 4. Class III cells.

Class III cells immunostained chromogenically for nestin are found densely distributed in the nucleus of the diagonal band (A) and medial septal nucleus (B). In the piriform cortex, Class III cells are lightly stained and found predominantly in layer II (C). Class III cells are scattered throughout the corpus striatum (D) and basal nucleus of Meynert (E), and are prominently displayed in the median preoptic nucleus (F) and the submamillothalamic nucleus of the hypothalamus (G). Class III cells found in the CA1, CA2, and CA3 fields of the hippocampus reveal a stereotyped structure consistent with that of pyramidal cells (H). Scale bar: 50 µm. Coronal drawings at three AP levels of the adult rat brain summarizing the regions in which NENs (black dots) are found and their relative distribution. The letters in each drawing correspond to the lettered panels A–H. NENs also are found in the islands of Calleja (IC), induseum griseum (IG), fasciola cinerea (FC) and preoptic nuclei (PN). Abbreviations: Corpus striatum (CS), hippocampal fields (CA1, CA2), medial septal nucleus (MSN), basal nucleus of Meynert (NBM), nucleus of the diagonal band (NDB), submamillothalamic nucleus (SMN).

Figure 5. Class III cells are NeuN⁺ nestin expressing neurons (NENs).

Confocal images of cells double stained for nestin (green) and neuronal nuclei (NeuN, red) reveal that all nestin⁺ cells in the regions described in Figure 4 also express NeuN, a marker associated exclusively with neurons. The cytological distribution of NeuN expression was identical in both NeuN⁺/nestin⁺ cells (NENs) and NeuN⁺/nestin^- cells. Many NeuN⁺/nestin^- nuclei are seen in close proximity to NeuN⁺/nestin⁺ cells. Shown are representative NeuN⁺/nestin⁺ NENs from the nucleus of the diagonal band (A), the medial septal nucleus (B), piriform cortex (C), corpus striatum (D), basal nucleus of Meynert (E), median preoptic nucleus (F), submamillothalamic nucleus (G), and CA2 field of the hippocampus (H). The asterisk in Figure 5G denotes the lumen of a blood vessel, cut in cross-section, surrounded by nestin⁺ endothelial cells, a configuration that could be mistaken for a nestin⁺ cell. Vertical images taken through the YZ plane of the tissue section confirm the co-localization of nestin and NeuN evident in the XY plane. Scale bar: 20 µm.

Figure 6. NENs express βIII-tubulin.

Confocal images of cells double-stained for nestin (green) and βIII-tubulin (red) demonstrate that all nestin⁺ cells in the regions described in Figures 4 and 5 also express βIII-tubulin, a cytoskeletal marker associated exclusively with neurons. The cytological distribution of βIII-tubulin expression was identical in both βIII-tubulin⁺/nestin⁺ cells (NENs) and βIII-tubulin⁺/nestin^- cells. Shown are representative βIII-tubulin⁺/nestin⁺ NENs from the nucleus of the diagonal band (A), the medial septal nucleus (B), piriform cortex (C), corpus striatum (D), basal nucleus of Meynert (E), median preoptic nucleus (F), submamillothalamic nucleus (G), and CA2 field of the hippocampus (H). Vertical images taken through the YZ plane of the tissue section confirm the co-localization of nestin and βIII-tubulin evident in the XY plane. Scale bar: 20 µm.

Figure 7. NENs express neurotransmitter-associated proteins.

Triple immunofluorescent staining of cells in the corpus striatum and in regions of the cholinergic basal forebrain confirm that all nestin⁺ cells (green) in these areas also express both NeuN (blue) and choline acetyltransferase (ChAT, red), a marker of cholinergic neurons. Representative cholinergic NENs in the medial septal nucleus (A), corpus striatum (B), and basal nucleus of Meynert (C) are shown. All ChAT⁺ cells, whether nestin⁺ or nestin^-, express NeuN. All nestin⁺ cells in the pyramidal cell layer of the hippocampus express both NeuN and the neuronal glutamate transporter EAAC1 (red), a marker of glutamatergic neurons. EAAC1⁺ NENs in all three hippocampal fields are structurally similar to the NEN shown in CA1 (D). Vertical images taken through the YZ plane of the tissue section confirm the co-localization of nestin and ChAT or nestin and EAAC1 evident in the XY plane of a given NEN. Scale bar: 20 µm.

Figure 8. Cell soma size of NENs.

Histograms showing the distribution of cell soma cross-sectional areas of NENs (black bars) and non-NENs (white bars) in the corpus striatum (A), the nucleus of the diagonal band (B) and the medial septal nucleus (C). In each of these regions, the mean cell soma size of NENs is significantly greater than the mean cell soma size of non-NENs (see Table 1), and NENs also are the largest cells in each region.

Figure 9. NENs are not recently born.

In adult rats given a single injection of BrdU (75 mg/kg) daily for 28 days and sacrificed 8 hours after the last injection, no NENs were labeled with BrdU, such as those seen in the CA1 field of the hippocampus (A), corpus striatum (B) and nucleus of the diagonal band (C). By contrast, BrdU⁺ cell nuclei were observed in each of these regions (arrowheads, A–C). In the granule cell layer of the dentate gyrus, some NeuN⁺ neurons displayed BrdU⁺ nuclei (arrow, D), whereas other BrdU⁺ nuclei were observed in cells that were NeuN⁻ (arrowhead, D). Vertical image taken through the YZ plane of the tissue section confirms the NeuN and BrdU co-localization evident in the XY plane. Scale bar: 20 µm (A–C), 30 µm (D).

Figure 10. Nestin expression by NENs does not protect against 192-Saporin cytotoxcity.

Cells in the nucleus of the diagonal band stained for choline acetyltransferase (ChAT) (A) and nestin (B). Cells expressing both ChAT and nestin appear yellow in the merged image (C). The number of nestin⁺/ChAT⁺ neurons is expressed as a percentage of the ChAT⁺ neurons in the nucleus of the diagonal band and medial septal nucleus in control rats, n = 3, and in rats that received an injection of the toxic ligand 192-Saporin at one of three doses, 0.5 µg, 1.0 µg, or 2 µg in 6 µL of PBS, n = 3, each dose, (D). Note that the percentage of nestin⁺/ChAT⁺ neurons remains essentially unchanged at each dosage of 192-Saporin, indicating that nestin expression does not afford any neuroprotection against the cytotoxcity induced by 192-Saporin. Scale bar: 50 µm.

Figure 11. NENs in the human forebrain.

NENs are present in a number of regions in the human forebrain, including the basal nucleus of Meynert (A), supraoptic nucleus (B), paraventricular hypothalamic nucleus (C), nucleus of the diagonal band (D), septal nucleus (E), lateral division of the bed nucleus of the stria terminalis (F), globus pallidus (G), and putamen (H). Scale bar: 100 µm (A, B, D, G) and 50 µm (C, E, F, H).

Figure 12. NENs in the human brain express choline acetyltransferase (ChAT).

NENs in the human forebrain express nestin (green) and ChAT (red) in the basal nucleus of Meynert (A), nucleus of the diagonal band (B), and putamen (C). Vertical images taken through the YZ plane of the tissue section confirm the co-localization of nestin and ChAT evident in the XY plane of a given NEN. Scale bar: 30 µm.