Fate of cajal-retzius neurons in the postnatal mouse neocortex - PubMed (original) (raw)

Fate of cajal-retzius neurons in the postnatal mouse neocortex

Tara G Chowdhury et al. Front Neuroanat. 2010.

Abstract

Cajal-Retzius (CR) neurons play a critical role in cortical neuronal migration, but their exact fate after the completion of neocortical lamination remains a mystery. Histological evidence has been unable to unequivocally determine whether these cells die or undergo a phenotypic transformation to become resident interneurons of Layer 1 in the adult neocortex. To determine their ultimate fate, we performed chronic in vivo two-photon imaging of identified CR neurons during postnatal development in mice that express the green fluorescent protein (GFP) under the control of the early B-cell factor 2 (Ebf2) promoter. We find that, after birth, virtually all CR neurons in mouse neocortex express Ebf2. Although postnatal CR neurons undergo dramatic morphological transformations, they do not migrate to deeper layers. Instead, their gradual disappearance from the cortex is due to apoptotic death during the second postnatal week. A small fraction of CR neurons present at birth survive into adulthood. We conclude that, in addition to orchestrating cortical layering, a subset of CR neurons must play other roles beyond the third postnatal week.

Keywords: Ebf2; apoptosis; cortical hem; green fluorescent protein; layer 1; marginal zone; reelin; two-photon.

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Figures

Figure 1

Figure 1

GFP+ cells in Ebf2 mice display the typical morphological features of Cajal–Retzius neurons. (A) Photomicrograph of the cranial window taken through the ocular of the two-photon microscope. A two-photon image of the boxed region is shown in panel (B). A, anterior; P, posterior; M, medial; L, lateral. (B) Low magnification two-photon image of GFP+ cells in somatosensory cortex of a P8 Ebf2-GFP mouse. The image is a composite maximum intensity projection (MIP) of six stacks, each consisting of 30–35 slices, 3 μm apart. An overlying dural vessel (red outline) partially obscures some of the fluorescence. (C) Higher magnification two-photon image (MIP of 57 slices, 2 μm apart) demonstrating that GFP-expressing cells have an oval cell body and a single (or sometimes two) main dendrite. The boxed region is shown in higher magnification in panel (E). (D) Side view (y_–_z projection) of the same image stack shown in panel (C) (scale bar also applies to (C)). Note that the main dendrite in CR neurons is oriented parallel to the pial surface with smaller secondary dendrites (arrows) that course up toward the pia (white arrows). Their axons are confined to layer 1 (not seen). (E) Higher magnification view of CR neuron dendritic spines in the boxed region in (B). This is a “best” projection of five optical slices, in which distracting processes (e.g., dendrites and axons of other CR neurons) have been digitally removed in Photoshop only for display purposes (Holtmaat et al., 2005). The morphological features shown in panels (B–E) define CR neurons.

Figure 2

Figure 2

GFP+ cells in Ebf2 mice express reelin. (A) Tangential sections cut through L1 of somatosensory cortex of fixed brains from Ebf2 mice at P2, P6 and P13, stained with antibodies to reelin (red), and then imaged with two-photon microscopy (green: native GFP fluorescence). (B) Quantitative analysis of the correspondence of immuno-reactivity for reelin and GFP expression throughout postnatal development. (C) Sections were also immuno-stained for GABA (red). GFP+ cells (arrowheads) do not express GABA; none of the GABA+ cells (white arrows) have a CR morphology. We also performed immunohistochemistry with anti-reelin and anti-GABA antibodies in wild-type mice and found that none of the reelin+ cells that were tadpole shaped express GABA (not shown).

Figure 3

Figure 3

GFP+ cells in Ebf2 mice display the typical electrophysiological properties of Cajal–Retzius neurons. (A) GFP+ cells in layer 1 of a P8 Ebf2 mouse were targeted for patch-clamp recordings and filled intracellularly with Alexa-594 (red) in acute coronal brain slices. (B) In the same slice, a GFP− cell was also targeted for recording; Inset: note that many thin dendrites (arrowheads) emanate radially from the rounder and larger soma of this cell (akin to the cells labeled by white arrows in Figures 2A,C). (C) Current clamp recording of a typical GFP+ cell showing broad action potentials with adaptation and a sag in the hyperpolarizing response, characteristic of CR neurons. (D) Current clamp recording of a fast spiking interneuron in L1. Current injection scale: −25, −15, −5, +5, +15, and +55 pA (C) or 255 pA (D).

Figure 4

Figure 4

Quantitative analysis of CR cell counts and disappearance rates in Ebf2 mice. (A) Time-lapse in vivo two-photon imaging of GFP+ CR neurons in a single Ebf2 mouse at P6, P8 and P12. In each animal we followed the fate of a large number of CR neurons in slightly overlapping stacks that we tiled together (due to the growth of the brain, the field of view underwent some degree of rotation and expansion over time). Seven representative CR neurons are indentified with numbers in all three images. (B) In this example, CR neurons were imaged chronically from P18 to P36. (C) Example of a mouse in which CR neurons were imaged chronically from P22 to P58. The images at the P36 (B) and P58 time points are dimmer because bone growth beneath the glass-covered window has partially obscured the neurons. Please note also that many CR neurons that survive into the second postnatal month tend to lose their principal dendrite and their cell bodies become rounder. (D) CR cell density throughout postnatal development. (E) Survival fraction (% CR neurons retained over subsequent imaging sessions) throughout postnatal development. Gray lines represent data from a single animal imaged chronically. Data for this graph came from a total of 1,660 CR neurons from 17 different mice imaged on at least two separate time points. (F) 2- or 4-day survival fractions across different postnatal ages. Animals/cells analyzed: three mice from P7 to P9 (437 cells), three mice from P11 to P13 (158 cells), three mice from P15 to P17 (62 cells), two mice from P18 to P22 (35 cells), five mice from P22 to P26 (86 cells), and two mice from P34 to P36 (36 cells). All images in panels (A–C) are MIPs of ∼28–35 slices, 3 μm apart.

Figure 5

Figure 5

Time-lapse imaging in Ebf-GFP mice confirms the death of identified CR neurons by apoptosis. (A) Serial imaging of an Ebf2-GFP mouse with in vivo two-photon microscopy to re-identify the location of individual CR neurons from P6 to P12. All images are MIPs of 30 slices, 2 μm apart. Note that CR neurons exhibit slight movements of the soma in the xy plane (blue arrow shows a large single day retraction of the soma of cell 13), but do not migrate over longer distances in the xy or z planes. Note also the disappearance of CR neurons 2, 3, 5, 7, 8, and 12 (red dashed circles). In many cases we “caught” the death of CR neurons by apoptosis (red arrows at P8 and P12). Some images were rotated slightly for alignment due to brain expansion throughout development. (B) Apoptosis of CR neurons occurs quickly (<6 h), as shown in this example at P15. Note that GFP expression persists until the time of the death. (C) Additional example of chronic in vivo two-photon imaging of apoptotic death of a CR neuron at P19-P10. Images are MIPs of 10–20 slices 2–3 μm apart.

Figure 6

Figure 6

Caspase-3 expression within dying CR neurons. Colocalization between activated caspase-3 (red) and Ebf2-GFP expression in dying CR neurons. Tangential sections through layer 1 of the neocortex of perfusion-fixed Ebf2-GFP mice at P8-P10 were stained with antibodies against activated caspase-3 and imaged with two-photon microscopy. Note how caspase-3 is expressed in the cytoplasm of dying CR neurons at the earliest stages of degeneration (top row), when CR neurons appear normal and maintain their tadpole morphology, as well as in late stages of cell death (bottom row), when only apoptotic bodies can be identified. Images are maximum intensity projections of 10–12 slices, 3 μm apart.

Figure 7

Figure 7

Retraction of CR neuron somata during early postnatal development. Left: in vivo two-photon time-lapse imaging of CR neurons in an Ebf2-GFP mouse from P6 to P10. A neuron at P7 (dashed circle) is seen undergoing apoptosis at P8 (red arrow). Right: same image sequence, in which four individual CR neurons are color-coded, to help visualize the retraction of cell bodies. Some somata (e.g., yellow cell) retract >100 μm.

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