Long-term channelrhodopsin-2 (ChR2) expression can induce abnormal axonal morphology and targeting in cerebral cortex

Abstract

Long-term expression of optogenetic proteins including channelrhodopsin-2 (ChR2) is widely used to study neural circuit function, but whether ChR2 expression itself perturbs circuits is not known. We expressed a common construct, CAG::ChR2 (H134R)-EYFP-WPRE, in L2/3 pyramidal cells in rat somatosensory cortex via in utero DNA electroporation (IUE). L2/3 pyramidal cells expressed ChR2-EYFP, but histology revealed abnormal morphology and targeting of ChR2-EYFP expressing axons, beginning at postnatal day (P) 33 and increasing with age. Axonal abnormalities included cylinders that enveloped pyramidal cell proximal apical dendrites, and spherical, calyx-like structures that surrounded neuronal cell bodies, including in L4. These are abnormal subcellular and laminar targets for L2/3 pyramidal cell synapses. Abnormalities did not occur in cells expressing GFP instead of ChR2, or in intermixed ChR2-negative axons. Long-term viral-mediated expression (80 d) did not cause axonal abnormalities when the CAG promoter was used, but produced some abnormalities with the stronger αCaMKII promoter (albeit much less than with in utero electroporation). Thus, under some circumstances high-level, long-term expression of ChR2-EYFP can perturb the structural organization of cortical circuits.

Keywords: axon development; circuit; in utero electroporation; optogenetics.

Figure 1

ChR2 expression in L2/3 pyramidal cells, as a function of age after in utero electroporation. (A) ChR2 expression in a P24 rat showing normal somatodendritic and axonal morphology of L2/3 pyramidal cells, including axon branches in L5. (B and C) Increasing appearance of abnormal EYFP+ puncta at P33 and P100. (D) Absence of puncta in a rat electroporated with GFP instead of ChR2 (age: P100). Scale bar, 200 μm. Numbers indicate cortical layers. wm, white matter.

Figure 2

Functional activation of ChR2 in vivo and in vitro. (A,B) Extracellular multiunit recording in L2/3 in vivo (200–300 um depth) in a urethane-anesthetized rat (P100). (A) Raw voltage recording at two time scales. (B) Spikes elicited by trains of 2-ms light pulses (473 nm, 20 mW, 50 Hz, 10 s intertrial interval). (C) Light-evoked potentials recorded from a ChR2-expressing L2/3 neuron in a brain slice, in response to increasing light intensity (2-ms light pulse).

Figure 3

Axonal swellings on ChR2+ axons. (A) Morphologically normal L2/3 pyramidal cell axons in L4 and L5 of electroporated S1, from a P100 rat electroporated with GFP only. (B–C) ChR2+ axons in L4 and L5 of two ChR2-electroporated rats, at P33 and P100. Arrowheads, cylindrical axonal swellings. Arrows, calyceal structures. (D–E) Axonal swellings in white matter underlying electroporated S1, and in callosal S1 axons in contralateral (non-electroporated) S1. (F) Dual fluorescent staining of ChR+ axons in L4 and the capillary marker collagen IV (magenta). Scale bar in (A–E), 50 μm. Scale bar in (F), 10 μm.

Figure 4

Absence of axonal swellings on intermixed ChR2-negative axons. (A) ChR2+ axons in S1 of P156 rat, showing numerous swellings. (B) Intermixed callosal axons from contralateral S1, anterogradely labeled by BDA injection and visualized with Alexa-647. Note absence of swellings. (C) Overlay. (D–E) Higher magnification of ChR2+ axons and BDA-labeled callosal axons. All images in (A–E) are maximum Z-projection confocal images. (F) Overlay of single confocal section of ChR2+ axons on top of image (E) of callosal axons. Scale bar is 500 μm for (in A for A–C) and 20 μm for (in D for D–F). Arrows show locations of ChR2-positive axonal swellings.

Figure 5

Axonal cylinders. (A–C) Example cylinders, showing patchy, mesh-like fluorescence. Arrowheads, single axon entering and exiting the cylinder. (D), Confocal sectioning of a cylinder, demonstrating the hollow, tubular structure. Scale bar for (A–D), 5 μm. (E–G), Some axonal cylinders surround pyramidal cell apical dendrites stained by SMI32 antibody. Panels show ChR2+ cylinders in layer 4, co-staining with SMI32 antibody, and overlay showing ChR2+ membrane surrounding dendrite (arrowheads). Insets in (G), single confocal optical sections showing ChR2 axon, SMI32, and overlay. Scale bar, 10 μm.

Figure 6

Calyx-like structures. (A–C), Example calyces. Scale bar, 5 μm. Small panels in (C) are confocal optical sections demonstrating hollow structure with entering axon (arrowhead). (D–F), Calyx structures surround neuronal somata. Panels show ChR2+ calyx, immunostaining for the neuronal marker Neu-N, and overlay. Scale bar, 10 μm.

Figure 7

Axonal swellings after viral-mediated expression of ChR2. (A,B), Low-power images of EYFP expression following long-term expression of AAV2.5-CAG-ChR2(H134R)-EYFP-WPRE (A; 80 days post-injection) and AAV2.5-αCaMKII-ChR2(H134R)-EYFP-WPRE [B(i) and B(ii), 80 and 100 days post-injection, respectively]. Arrows in (A), infected astrocytes. Arrowheads in (B), ChR2 puncta. Scale bar for (A,B) is 500 μm. (C) Examples of axonal malformations in AAV2.5-αCaMKII cases. Arrowhead in C(iv), entering/exiting axon. Scale bar, 10 μm.

Figure 8

ChR2 puncta density correlates with ChR2 expression level. (A), IUE cases. Each point represents one animal, analyzed in L2/3 at region of maximal S1 expression. Color indicates age. ChR2 expression is estimated from EYFP intensity, defined as the mean brightness value (range: 0–255) for the brightest 20% of pixels in the analysis region. Line is linear regression (n = 14 cases). With the exception of the youngest ages (P24–33), puncta density correlated strongly with ChR2 expression level. (B) AAV-CaMKII-ChR2 cases (n = 10 animals). Color indicates days post-injection (dpi).