New neurons in neocortex
Based on the premise that new cortical neurons would migrate from the SVZ across the subcortical white matter, we examined BrdU-immunoreactive (+) cells in the deep portion of the rostral neocortex (Bregma 1.20–3.20 mm; Paxinos and Watson, 1998
) at several time points after BrdU injection (). Examination of brain sections from rats injected with BrdU 4–5 wk earlier revealed BrdU+ cells that were double labeled with antibodies against each of three neuronal markers: NeuN, a marker specific for mature neurons; EAAC-1, a neuronal glutamate transporter; and HuC/D, neuron-specific RNA binding proteins (; and ). BrdU+ cells double labeled with neuron-specific enolase (NSE), a marker of metabolically active neurons (Marangos and Schmechel, 1987
), were found only at a later time point, 11–12 wk after BrdU injection (). BrdU+/NeuN+ neurons were also observed at this longer survival time ().
BrdU injections and survival times
Figure 1. Some cells born in the adult cortex express neuronal markers. (A) A 4–5-wk-old cell labeled with BrdU and the mature neuronal marker NeuN is shown within the green box in A1. Sample z-planes through the boxed area in A1 are shown with color separation (more ...)
Immunostaining of BrdU-labeled cells in the adult rat neocortex and striatum
Antibodies used for immunohistochemistry
Systematic quantitative analysis of the deepest 500 μm of the rostral neocortex (comprising layer VI and in some areas part of layer V) was done in one half section (Bregma 1.2 mm) from each of 10 rats injected 4–5 wk earlier. This analysis revealed seven BrdU+ cells that were also NeuN+ (4.9 ± 2.9 cells/mm3
; 0.6 ± 0.3% of 798 BrdU+ cells, both mean ± SEM), all with NeuN staining comparable to that of neighboring neurons. To look for additional double-labeled cells using a less time-consuming method of analysis, an additional 3.5 sections (Bregma 1.2–3.2) from each of the 10 rats were analyzed by scanning on the confocal microscope only those BrdU+ cells that appeared to be NeuN+ under epifluorescence. This second scan resulted in 26 cells BrdU+/NeuN+ cells (2.8 ± 0.9 cells/mm3
; 0.4 ± 0.1% of 6826 BrdU+ cells), for an overall density of 3.1 ± 1.1 cells/mm3
. Variability was seen across rats: 3 of the 10 brains analyzed contained no BrdU+/NeuN+ cells in any of the four sections sampled, whereas one brain contained as many as 11 BrdU+/NeuN+ cells in four sections. High variability is also seen across individuals in the dentate gyrus (Dayer et al., 2003
), where it is likely to reflect a combination of genetic and environmentally induced differences in rates of neurogenesis (Kempermann et al., 1997a
). BrdU+/NeuN+ cells were observed within the infralimbic cortex, cingulate cortex, somatosensory cortex, secondary motor cortex, and claustrum. No double-labeled cells were seen in the intervening primary motor cortex, which comprised ~20% of the region analyzed.
Because of their small size and lack of cytoplasmic NeuN staining, none of the cells double labeled with BrdU and neuronal markers had the appearance of pyramidal neurons. All had small nuclei, 5–10 μm in diameter, similar in size to newborn neurons previously found in the adult mammalian cortex (Kaplan, 1981
; Magavi et al., 2000
; Gould et al., 2001
). The 33 BrdU+/NeuN+ cells counted above had a mean nuclear diameter of 7.6 ± 0.2 μm, and five BrdU+/NSE+ cells had nuclei measuring 7.4 ± 0.7 μm. This small size suggested that newborn neurons in the neocortex were interneurons, a possibility that was examined by immunostaining for the neurotransmitter γ-aminobutyric acid (GABA). A small fraction of 4–5-wk-old BrdU+ cells were GABA+ (). Because GABA immunoreactivity could potentially result from uptake rather than production of GABA, sections were also stained for the GABA synthesizing enzyme glutamic acid decarboxylase 67 (GAD-67). No BrdU+/GAD67+ cells were found in the deep layers of the neocortex 4–5 wk after BrdU injection. However, several BrdU+/GAD67+ cells were encountered at the 11–12 wk time point (). Why 4–5-wk-old cells would label for GABA and not for GAD is unclear; however, strong GABA immunoreactivity and weaker GAD67 expression have previously been observed in small cortical neurons in the adult primate, where this pattern was thought to result from relatively low activity levels suggesting immature neurons (Hendrickson et al., 1994
). All of the BrdU+/GABA+ and BrdU+/GAD67+ cells also had small nuclei, 7.4 ± 0.3 μm and 7.3 ± 0.3 μm in diameter, respectively. Calcium binding protein expression was examined, because functional classes of GABAergic interneurons are often identified using these markers (Gabbott and Bacon, 1996
; Gabbott et al., 1997
). Calbindin (CB) and calretinin (CR) immunoreactivity were each observed in 4–5-wk-old and 11–12-wk-old BrdU+ cells (). In triple-labeled sections, no BrdU+/CR+ cells appeared to be CB+, suggesting that there are two distinct classes of newly generated cortical interneurons or, alternatively, that new interneurons make a developmental switch from CR to CB expression like the adult-born dentate gyrus granule neurons (Brandt et al., 2003
). BrdU+/CR+ and BrdU+/CB+ cells also had small nuclear diameters (7.0 ± 0.3) like cells labeled with other neuronal markers. No 4–5-wk-old BrdU+/parvalbumin+ cells or BrdU+/somatostatin+ cells were found.
Figure 2. Some cells born in the adult cortex express markers of GABAergic interneurons. (A) A 4–5-wk-old BrdU+/GABA+ cell is shown within the boxed area in the multi-channel view in A1 and at higher magnification in A2. (B) An 11–12-wk-old BrdU+/GABA+ (more ...)
Figure 3. Some cells born in the adult cortex express CB or CR, markers of specific interneuron classes. (A) A pair of 4–5-wk-old BrdU+/CB+ neurons is shown within the box in A1 and at higher magnification color separation in A2. Pairs of BrdU+ labeled (more ...)
Because of their close apposition to other, generally larger, cells ( and ), some BrdU+/GABA+, BrdU+/GAD67+, BrdU+/CB+, and BrdU+/CR+ cells appeared to be “satellite cells”. This suggests that some satellite cells, previously described as cells that could be mistaken for the nuclei of BrdU-labeled pyramidal cells (Gould et al., 1999b
; Kornack and Rakic, 2001
), may actually be newly born GABAergic neurons themselves. If this is the case, BrdU+/NeuN+ cells may have been undercounted in the quantitative analysis, because all satellite cells were classified as NeuN-nonimmunoreactive (NeuN−) in this analysis, due to the difficulty in determining the NeuN immunoreactivity of a small cell in very close proximity to a large NeuN+ cell. Even using high resolution Z-sectioning, NeuN immunoreactivity from the large neuron precludes unambiguous scoring of NeuN expression in a closely apposed BrdU+ satellite cell ( A).
Figure 4. Many cells in the adult rat cortex do not appear to be neurons or commonly recognized glial types. (A) BrdU+ satellite cells closely apposed to large neurons could lead to erroneous identification of the large neurons as BrdU labeled. However, Hoechst (more ...)
Source of new neocortical neurons
To investigate whether new cortical neurons arise from precursors in the SVZ, coronal sections through the rostral forebrain (Bregma 0.48–2.20 mm) were immunolabeled for BrdU and the immature neuronal marker doublecortin (DCX). Several isolated BrdU+/DCX+ cells were observed within the subcortical white matter of the forceps minor and external capsule (, A–C). These cells had the appearance of migrating young neurons, with elongated cell bodies and one or two long leading processes directed away from the SVZ. Triple staining for BrdU, DCX, and the immature neuronal marker CRMP4 (sometimes referred to as TOAD-64 or TUC-4) revealed that all BrdU+/DCX+ cells were also CRMP4+ ( C). Surprisingly, no DCX+ cells, with or without BrdU labeling, were observed within the deep layers of the neocortex in 48 sections from 12 brains. However, a small number of 1–4-wk-old BrdU+/CRMP4+ cells that were DCX- were observed in this region ( D). It is possible that BrdU+/DCX+ cells enter from the subcortical white matter and down-regulate DCX as soon as they do so. However, the lack of DCX immunoreactivity in BrdU+/CRMP4+ cells, along with the observation that BrdU+/NeuN+ cells, BrdU+/CB+ cells, and BrdU+/CR+ cells often occurred in pairs <25 μm apart (4 of 33 cells, 2 of 5 cells, and 6 of 10 cells, respectively), suggested that new cortical neurons might arise from in situ progenitors rather than from the SVZ.
Figure 5. Young cells in the adult cortex expressing CRMP4, NG2, and NeuN, but not DCX, suggest in situ neurogenesis. (A–C) Isolated 1–2-wk-old BrdU+/DCX+ and BrdU+/DCX+/CRMP4+ neurons in the subcortical white matter (wm) dorsal to the striatum (more ...)
To explore this possibility, the identity of proliferating cells in the deep cortical layers was examined 2 h after BrdU injection. 92.4% ± 3.8 of 152 BrdU+ cells scanned in the deepest 500 μm of the neocortex had cell bodies and processes that were immunoreactive for the chondroitin sulfate proteoglycan NG2. The majority of BrdU+/NG2− cells could be identified as endothelial cells based on their distinctive morphology, i.e., a flattened and curved shape consistent with wrapping around a blood vessel (Palmer et al., 2000
). None of the BrdU+ neocortical cells scanned at this 2 h time point were NeuN+. However, at the 4–5 wk survival time point, processes with faint to moderate NG2 immunoreactivity were observed in 2 of the 7 BrdU+/NeuN+ cells in one sample and in 9 of the 26 BrdU+/NeuN+ cells in the second sample (, E–G). NeuN+/NG2+ cells were also found in sections not stained for BrdU, indicating that colabeling was not an artifact of BrdU staining ( H). In rare cases, 11–12-wk-old BrdU+/NeuN+ neurons still displayed faint NG2 immunoreactivity.
Nearly 60% of 4–5-wk-old BrdU+ cells were NG2+/NeuN−. Many of these cells appeared to be satellites of large neurons (), similar to some of the BrdU+ cells labeled with neuronal markers. NG2+ cells were further characterized using several glial markers (). A recent study showed NG2 staining of EGFP+ cells in S100β-EGFP expressing transgenic mice (Vives et al., 2003
). However, we found no NG2+/S100β+ cells among 130 NG2+ and 118 S100β+ cells. Triple labeling for BrdU, NG2, and either GFAP or CNPase showed that none of the BrdU+/NG2+ cells expressed these astrocyte or oligodendrocyte markers. A few cells were double labeled with BrdU and either CNPase, S100β, or the microglial marker Iba-1, but the number of these new glial cells was low and could not account for the ~40% of BrdU+ cells that were NG2− and NeuN− in the systematic analysis (Table II; D).
New neurons in the striatum
To investigate the fate of BrdU+/DCX+ cells in the SVZ that do not follow the rostral migratory stream and do not appear to enter the cortex, we examined the region surrounding the SVZ 1–2 wk after BrdU injection. A large number of 1–2-wk-old BrdU+/DCX+ cells were observed in the portion of the SVZ stretching laterally from the dorsolateral corner of the lateral ventricle, between the striatum and corpus callosum (). Examination of the striatum revealed BrdU+/DCX+/CRMP4+ cells whose location and morphology suggested that they could be migrating within the fiber tracts that course through the striatum (). A few BrdU+/DCX+/CRMP4+ cells were also found within the striatal matrix, i.e., the portion of the striatum outside the fiber tracts, at this time point. 2 wk later (3–4 wk after BrdU injection), BrdU+/DCX+ cells had more branched processes (). BrdU+/DCX+/NeuN+ neurons were also found in both the dorsomedial striatum and nucleus accumbens at this time point (, G–I). 4–5 wk after BrdU injection, BrdU+/NeuN+ cells in the striatum had rounder nuclei than BrdU+/NeuN+ cells observed at 3–4 wk ( A). 11–12 wk after BrdU injection, BrdU+/NSE+ as well as BrdU+/NeuN+ cells were found in the striatum (, B–D).
Figure 6. Young neurons appear to migrate from the adult SVZ into the striatum. (A and B) A band of DCX+ and CRMP4+ 1–2-wk-old BrdU+ neurons appears in the extension of the SVZ between the striatum (str) and the corpus callosum (cc). Scattered immature (more ...)
Figure 7. BrdU-labeled cells in the adult striatum express markers of GABAergic interneurons. (A) A 4–5-wk-old BrdU+ cell expressing the neuronal marker NeuN in the striatum just outside a striatal fiber tract (sft) is seen in the box in A1, in color separations (more ...)
BrdU+/GAD67+ and BrdU+/CR+ cells were both observed 4–5 wk after BrdU injection (, E–H), suggesting that newborn neurons in the striatum were GABAergic interneurons as in the neocortex. No double labeling of 4–5-wk-old BrdU+ cells was found with CB, parvalbumin, or somatostatin.
The vast majority of the new neurons in the striatum, identified with NeuN, GAD-67, or CR, were found either in the nucleus accumbens or within a restricted dorsomedial region of the striatum, within 650 μm of the lateral edge of the SVZ, ~10–15 times the width of the SVZ ( H, inset). This portion of the striatum has been found to contain large numbers of CR+ interneurons and is believed to be an associative region of the striatum, corresponding to the caudate region in nonrodents (Riedel et al., 2002
). 36.4 ± 7.0 BrdU+/NeuN+ cells/mm3
(5.8 ± 0.9% of 995 BrdU+ cells) were counted in the dorsomedial striatum at this time point. The location, immunoreactivity, and nuclear diameter (5–10 μm) of these BrdU+ cells were all consistent with striatal CR+ interneurons (Riedel et al., 2002