Genetically-directed, cell type-specific sparse labeling for the analysis of neuronal morphology

Abstract

Background: In mammals, genetically-directed cell labeling technologies have not yet been applied to the morphologic analysis of neurons with very large and complex arbors, an application that requires extremely sparse labeling and that is only rendered practical by limiting the labeled population to one or a few predetermined neuronal subtypes.

Methods and findings: In the present study we have addressed this application by using CreER technology to non-invasively label very small numbers of neurons so that their morphologies can be fully visualized. Four lines of IRES-CreER knock-in mice were constructed to permit labeling selectively in cholinergic or catecholaminergic neurons [choline acetyltransferase (ChAT)-IRES-CreER or tyrosine hydroxylase (TH)-IRES-CreER], predominantly in projection neurons [neurofilament light chain (NFL)-IRES-CreER], or broadly in neurons and some glia [vesicle-associated membrane protein2 (VAMP2)-IRES-CreER]. When crossed to the Z/AP reporter and exposed to 4-hydroxytamoxifen in the early postnatal period, the number of neurons expressing the human placental alkaline phosphatase reporter can be reproducibly lowered to fewer than 50 per brain. Sparse Cre-mediated recombination in ChAT-IRES-CreER;Z/AP mice shows the full axonal and dendritic arbors of individual forebrain cholinergic neurons, the first time that the complete morphologies of these very large neurons have been revealed in any species.

Conclusions: Sparse genetically-directed, cell type-specific neuronal labeling with IRES-creER lines should prove useful for studying a wide variety of questions in neuronal development and disease.

Figure 1. Restriction maps of the targeting constructs, targeted alleles, Southern blot probes, and PCR primers for IRES-CreER knock-in alleles at the ChAT, NFL, TH, and VAMP2 loci.

For each knock-in allele, a cassette consisting of an IRES-CreER, the pBR322 Tetracycline resistance gene (TetR), and an Frt-flanked phosphoglycerate kinase promotor neomycin resistance gene (FNF) was inserted into the 3′ untranslated region of the target gene as indicated. The PGK-neo casette was subsequently removed in vivo by Flp recombinase. The sizes of the homology regions used for the targeting constructs were: ChAT, 9.5 kb; NFL, 7.0 kb; TH, 9.2 kb; and VAMP2, 9.1 kb. For Southern blot hybridization, the probes indicated beneath the maps of the targeted loci were used, and genomic DNAs from the four groups of targeted ES cells and germ-line transmitted mice were cleaved with the following restriction enzymes: ChAT, Stu I and BamH I; NFL, EcoR V; TH, BamH I; and VAMP2, Nco I.

Figure 2. Cell-type specificity of Cre recombinase activity in mouse lines with different IRES-CreER knock-ins.

The cell-type specificity of each line was tested by crossing to the Z/AP reporter and inducing sparse recombination with 4HT. A, In the retina, the NFL-IRES-CreER line exhibits Cre-mediated recombination in retinal ganglion cells, each of which has a single axon projecting to the optic disc, and in horizontal cells; the image shown here shows only retinal ganglion cells. B, in the brain, the VAMP2-IRES-CreER line exhibits Cre-mediated recombination in both neurons and glia. C and D, in the retina, the ChAT-IRES-CreER line exhibits Cre-mediated recombination exclusively in cholinergic (starburst) amacrine cells. The region boxed in C is shown at higher magnification in D. Scale bars: 0.5 mm in A and B; 1 mm in C.

Figure 3. Sparse recombination permits visualization of non-overlapping neuronal processes.

A, 200 um vibratome section from the brain of an NFL-IRES-CreER;Z/AP mouse injected at P0 with 0.2 mg 4HT. In this section, three large neurons (or parts of neurons) are labeled in one hemisphere of the cerebral cortex. B–D, enlarged bright field images at one Z-plane for each of the three neurons. Scale bar: 0.5 mm.

Figure 4. Tracing the morphologies of diverse AP expressing neurons from thick coronal sections of adult brains from various IRES-CreER;Z/AP mice.

A, Multipolar neuron in the inferior cerebral cortex. B, Mossy fibers in the cerebellum. C, The cortical pyramidal cell shown in Figure 3B. D, Cerebellar granule cells. E, a compact cortical pyramidal cell. A–D are from NFL-IRES-CreER;Z/AP brains, and E is from a ChAT-IRES-CreER;Z/AP brain; the tracing of the cell in E is shown at lower magnification in Figure 5J. A, B, D, and E are traced from 300 um thick sections, and samples from the corresponding Z-series bright field images are shown. C is traced from a 200 um thick section; a sample from the Z-series image is shown in Figure 3B. Scale bars: 0.2 mm.

Figure 5. Morphologies of cholinergic neurons in an adult ChAT-IRES-CreER;Z/AP brain.

Different neurons are color-coded. The mouse received injections of 4HT on P8 (1 mg), P21 (2 mg), and P28 (2 mg). A–P, tracing of neuronal processes from 16 serial 300 um coronal sections. Q–S and Figure 4E show bright field images of the AP-stained tissue corresponding to the boxed and lettered regions in D and J. All of the AP-stained neurons within the 16 sections are shown. Regions in which a high density of AP-labeled processes precluded accurate tracing are indicated by a semi-opaque grey zone, as shown for the cell in panel R. Cells bodies are indicated by black arrows. Scale bars: 0.2 mm in Q and R, 0.5 mm in S.

Figure 6. Morphologies of AP-labeled neurons in the rostral half of an adult ChAT-IRES-CreER;Z/AP brain.

The mouse received injections of 4HT on P8 (1 mg), P21 (2 mg), and P28 (2 mg). A,B, bright field images of AP-stained tissue corresponding to the boxed regions in J and E, respectively. C–M, AP-labeled processes traced from eleven serial 300 um coronal sections. Two adjacent neurons with cell bodies in the basal forebrain (black arrows in panels K and L) and their large axon arbors in the cerebral cortex are shown in red. All other processes are shown in green. Regions in which a high density of AP-labeled processes precluded accurate tracing are marked by semi-opaque grey zones. Scale bar: 0.5 mm in A, 0.2 mm in B.

Figure 7. Tracing the morphology of a single AP-expressing neuron projecting from the periaqueductal gray to the lateral hypothalamus in an adult TH-IRES-CreER;Z/AP brain.

A–O, Fifteen serial 300 um coronal sections. This is the only AP-expressing neuron in this hemisphere. The cell body is indicated by the black arrow in N.

Figure 8. AP-labeled processes in the hippocampus in an adult NFL-IRES-CreER;Z/AP brain.

A–L, Contiguous AP-labeled processes span eleven serial 300 um sections within the hippocampus. All of the AP labeled processes within the two hippocampi have been traced, but AP-labeled neurons outside of the hippocampus are not shown. M, anterior hippocampus corresponding to the boxed region in B shows unilateral AP-labeling. Some residual endogenous alkaline phosphatase activity is seen in the vasculature and choroid plexus. The cell body of this neuron has not been localized. N,O, Two images from a Z-stack of the region bounded by the pair of black corner marks in M. The processes of the AP-labeled neuron form a finely spaced meshwork near the surface of the molecular layer of the dentate gyrus. Scale bars: 0.5 mm in M, 0.2 mm in O.