Origins of cortical interneuron subtypes - PubMed (original) (raw)

Origins of cortical interneuron subtypes

Qing Xu et al. J Neurosci. 2004.

Abstract

Cerebral cortical functions are conducted by two general classes of neurons: glutamatergic projection neurons and GABAergic interneurons. Distinct interneuron subtypes serve distinct roles in modulating cortical activity and can be differentially affected in cortical diseases, but little is known about the mechanisms for generating their diversity. Recent evidence suggests that many cortical interneurons originate within the subcortical telencephalon and then migrate tangentially into the overlying cortex. To test the hypothesis that distinct interneuron subtypes are derived from distinct telencephalic subdivisions, we have used an in vitro assay to assess the developmental potential of subregions of the telencephalic proliferative zone (PZ) to give rise to neurochemically defined interneuron subgroups. PZ cells from GFP+ donor mouse embryos were transplanted onto neonatal cortical feeder cells and assessed for their ability to generate specific interneuron subtypes. Our results suggest that the parvalbumin- and the somatostatin-expressing interneuron subgroups originate primarily within the medial ganglionic eminence (MGE) of the subcortical telencephalon, whereas the calretinin-expressing interneurons appear to derive mainly from the caudal ganglionic eminence (CGE). These results are supported by findings from primary cultures of cortex from Nkx2.1 mutants, in which normal MGE fails to form but in which the CGE is less affected. In these cultures, parvalbumin- and somatostatin-expressing cells are absent, although calretinin-expressing interneurons are present. Interestingly, calretinin-expressing bipolar interneurons were nearly absent from cortical cultures of Dlx1/2 mutants. By establishing spatial differences in the origins of interneuron subtypes, these studies lay the groundwork for elucidating the molecular bases for their distinct differentiation pathways.

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Figures

Figure 1.

Figure 1.

Absence of somatostatin- and parvalbumin-expressing interneurons in primary cultures of Nkx2.1_–/_– cortex. Cortices from E18.5 embryos (top panels, +/+; bottom panels, –/–) were dissociated, and cells were plated at high density and cultured for 2–4 weeks before fixation and immunolabeling. Despite the presence of background staining, parvalbumin (Parv)-, somatostatin (Somt)-, and NPY-expressing cells were absent in the mutant cultures; however, calretinin-expressing (Calr) cells appeared normal. These results correlate well with the transplantation studies described below in Results, which suggest that interneuron subtypes have distinct origins. Scale bar, 100 μm.

Figure 7.

Figure 7.

Birth dating of calretinin-expressing cortical interneurons. A–D, Low-magnification views of BrdU staining in the somatosensory cortex of animals that received five injections of BrdU (50 mg/kg) 2 hr apart at E12.5 (A), E14.5 (B), E16.5 (D), or E18.5 (D). E, F, High-magnification views of calretinin (red) and BrdU (green) labeling in layer II from animals that received E12.5 (E), E14.5 (F), or E16.5 (G) injections. Most of the calretinin-expressing interneurons have a bipolar morphology similar to that seen in vitro and are labeled by BrdU injections at E12.5 (79%) or E14.5 (70%) but rarely later (only 1.8% at E16.5, and essentially none from injections at E18.5 or P1). Scale bar, 20 μm.

Figure 8.

Figure 8.

Transplanted progenitors from the E14.5 CGE give rise to calretinin-expressing cells. A, The region of CGE that was dissected from a 300-μm-thick coronal section at E14.5. This region is dorsal to the Nkx2.1-expressing domain (Nery et al., 2002). Many of the GFP plus CGE donor cells (B) colabel for calretinin (C). These colabeled neurons do not also label for reelin (D) (reelin triple labeled with GFP and calretinin in a total of 6 of 201 cells from 3 separate experiments). Graph in E shows that CGE donors generate calretinin-expressing (Calr) cells primarily at E14.5 and that relatively few parvalbumin (Parv)- or somatostatin (Somt)-expressing neurons are generated by these transplants. F, Many of the calretinin+ donor neurons from CGE were generated by progenitors that proliferated after transplantation. The graph shows the percentage of GFP+–calretinin+ neurons from the E14.5 CGE that proliferated (triple labeled with BrdU) during the first, second, or third DIV. The rise in triple-labeled cells between the first and second DIV results from the addition of FGF2 to the medium at the start of 2DIV as indicated by arrowheads. The graph in G shows BrdU incorporation by GFP+ donor neurons from the MGE at E12.5 and from the MGE, LGE, or CGE at E14.5. Despite high levels of proliferation during the first day in vitro for MGE donors at E12.5 and modest initial levels of proliferation for MGE or LGE donors at E14.5, only the CGE donors give rise to large numbers of calretinin+ neurons. Ctx, Cortex, Th, thalamus; Hyp, hypothalamus; Calr, calretinin; Rln, reelin. Scale bars: A, 500 μm; B_–_D, 50 μm.

Figure 9.

Figure 9.

Differential loss of bipolar calretinin-expressing neurons in Dlx1/2_–/_– cortical cultures. A, Cultures of dissociates cortex from E18.5 wild-type embryos produce large numbers of calretinin-expressing cells, few of which colabel with reelin (B), a marker of Cajal-Retzius cells. C, Cultures from Dlx1/2 mutants show a large reduction of calretinin-labeled cells. D, Nearly all of the remaining calretinin+ cells from the mutant cultures strongly express reelin and have a large unipolar morphology that is suggestive of a Cajal-Retzius cell (Pappas and Parnavelas, 1998) (quantified in F; there were significantly lower percentages of bipolar and higher percentages of unipolar calretinin+ cells in the Dlx1/2_–/_–; p < 0.0001). This nearly complete loss of small bipolar reelin-negative calretinin-expressing cells stands in marked contrast to the more modest reductions of somatostatin- or NPY-expressing interneurons (quantified in E). Scale bar, 100 μm. *p < 0.05; paired t test.

Figure 2.

Figure 2.

Differential expression of GABA or glutamate in donor cells from the proliferative zone of the MGE or cortex. In this experiment the MGE or neocortex of E14.5 donors was plated onto neonatal cortex feeders and cultured for 2 weeks. Each row of three panels shows identical fields: the left row (B, E, H, K) shows GFP expression by the donor cells, the middle row shows expression of glutamate (C, I) or GABA (F, L), and the right row shows the merged images. Few donors from the MGE (B_–_D) express high levels of glutamate, whereas many more express GABA (E_–_G). In contrast, most cortical donors (H_–_J) strongly express glutamate, whereas few express high levels of GABA (K_–_M). Scale bar, 50 μm.

Figure 3.

Figure 3.

GABA and glutamate expression by transplanted donors from the MGE, LGE, or neocortex after 14 DIV. The percentages of GFP+ donor cells with neuronal morphology and that also label for GABA or glutamate are plotted for the three progenitor domains dissected early (E12.5), mid (E14.5), and late (E16.5) in cortical neurogenesis. *p < 0.04; paired t test.

Figure 5.

Figure 5.

Parvalbumin, somatostatin, and calretinin expression by transplanted proliferative zone cells from the MGE, LGE, or neocortex. Parvalbumin labeling was examined after 28 DIV; somatostatin and calretinin labeling was examined after 14 DIV. The percentages of GFP+ donor cells with neuronal morphology and that also label for these markers of interneuron subgroups are plotted for progenitor domains dissected early (E12.5), mid (E14.5), and late (E16.5) in cortical neurogenesis. Statistical analysis by ANOVA revealed significant effects of area on the percentage of transplanted neurons expressing parvalbumin (p < 0.003 for each age) and somatostatin (p < 0.001 at E14.5 only).

Figure 4.

Figure 4.

Transplanted progenitors from the MGE give rise to distinct interneuron subtypes. In this experiment the E14.5 MGE donor cells were plated onto neonatal cortex feeders and cultured for 2 weeks (4 weeks in the case of parvalbumin). Each set of three panels shows identical fields, the left side of which shows GFP expression by the donor cells. The middle panels show expression of the interneuron subtype markers parvalbumin (Parv; B), somatostatin (Somt; E), neuropeptide Y (NPY; H), and calretinin (Calr; K). The panels on the right side show the merged images. Colabeling is present between some of the MGE donor cells and Parv, Somt, and NPY; however, MGE donors do not colabel for calretinin. Scale bar, 50 μm.

Figure 6.

Figure 6.

DARPP32 expression in neurons from E14.5 LGE but not MGE. A, B, The same field from an experiment in which LGE donors from E14.5 embryos were cultured for 1 week on a cortical feeder layer. The only DARPP32+ cell (arrow) in A is a LGE donor cell as indicated by colabeling for DARPP32 and GFP in B. C, LGE cells cultured on striatal feeders can also express DARPP32 (arrow). In contrast to cells from the LGE, no MGE donor cells express DARPP32 when cultured on cortex (as in D) or on striatum (data not shown).

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