Figure 1.
Cre-activated AAV vectors confer cell-type specific and high-level gene expression in brains of Cre knockin mice.
(a) Injection of a generic AAV-GFP virus into neocortex of a Pv-cre mouse labeled neurons non-specifically. Pyramids can be recognized by the presence of thick apical dendrites (black arrowheads) and projecting axons (white arrows). (b) Injection of the AAV-lox-STOP-lox (LS1L)-GFP virus into the neocortex of a Pv-cre mouse specifically labeled neurons expressing Cre-recombinase. The STOP cassette is shown in gray in the schematic and is described in detail in Methods. Δβgeo-3xpA, a modified beta-galactosidae/neomycin STOP cassette with 3 poly-adenylation sites. (c) Injection of the AAV-LS2L-RFP virus into the neocortex of a Pv-cre mouse specifically labeled neurons expressing Cre-recombinase. A different version of STOP cassette is used here, Neo-2xpA, a neomycin STOP cassette with 2 poly-adenylation sites. PCMV, CMV promoter and β-globin intron; scale bar, 250 µm for a–c. (d) Co-localization (white arrowheads) of GFP and parvalbumin (PV) in neocortical basket interneurons in neocortex of a Pv-cre mouse injected with AAV-LS1L-GFP; scale bar, 20 µm. Far right, high resolution image of basket cell axons with “basket-like” terminal branches and boutons (yellow arrowheads) around pyramidal cell somata (labeled with NeuN immunofluorescence) characteristic to PV+ interneurons; scale bar, 5 µm. (e) Co-localization of GFP and LacZ in neocortical pyramidal neurons in a Emx-cre-nlsLacZ mouse injected with AAV-LS1L-GFP; scale bar, 20 µm. Note that not all LacZ+ pyramidal neurons were positive for GFP. Far right, spines (blue arrowheads) along a pyramidal cell apical dendrite; scale bar, 5 µm. (f) Quantification of GFP fluorescence intensity at the soma normalized to intensity of GFP-labeled pyramidal soma in Thy1-GFP mice; n = 15 cells for each group. The intensity range for both Thy1-GFP and AAV-labeled somata was large, only the brightest cells were selected for this particular comparison (see Methods for details). Thy1-GFP: 1+/−0.08; AAV-LS1L-GFP::Pv-cre: 1.5+/−0.1; AAV-LS1L-GFP:: Emx-cre 1.3+/−0.2). All AAV-injected tissue was fixed 12–15 days post injection of 1.5–3 month-old mice.
Figure 2.
GFP expression in Pv-cre mice injected with AAV-LS1L-GFP is stable for months.
(a) Pv-cre mice (top row; n = 3 mice for each time point) and wildtype control mice (middle row; n = 3 mice for each time point) were injected with AAV-LS1L-GFP and were perfused at 6,15,30, and 60 days post-injection (dpi). See Figure S3 for 60 dpi. All epi-fluorescence images were acquired with a CCD camera using the same acquisition parameters. The maximum exposure time that did not lead to saturation of signal was used (75 ms), and the same look-up table was applied to all of the images shown. GFP expression levels increased with time in Pv-cre mice. Under these conditions, sparse weak signals were detected in some wildtype control mice (blue arrowheads); scale bar, 100 µm. Bottom row, quantification of GFP intensity at somata of labeled neurons plotted for wildtype control mice (blue) and Pv-cre mice (black). Average number of GFP+ cells counted per animal in Pv-cre injected animals was (6,15,30 dpi): 110+/−34, 215.3+/−34, 195.3+/−26 cells. (b) Whole cell current clamp recording of an AAV-LS1L-GFP-transfected neuron from a 2.5 month-old Pv-cre mouse at 20 dpi. Left, DIC image of recorded cell; inset shows GFP fluorescence; scale bar, 10 µm. Right, example trace of spikes in response to a 250 pA current step; scale bar, 10 mV, 25 ms.
Figure 3.
Functional demonstration that the Cre-activated AAV vector is cell-type specific.
Pv-cre mice were injected with AAV-LS2L-ChR2mCherry and acute slices for electrophysiological recording were prepared. (a–c) Light stimulation directly elicited depolarizing responses in ChR2-positive neurons. DIC and fluorescent images of a ChR2-positive cell targeted for recording (a); scale bar: 10 µm. Spike responses to light stimulation of increasing duration (7, 14, and 500 ms as indicated by blue line) were recorded in cell-attached mode (b); scale bars: 40 pA, 25 ms. ChR2-mediated current was maintained in the presence of TTX. (c); scale bar: 50 pA, 10 ms. (d) Electrical field stimulation evoked both inhibitory post-synaptic current (upper trace, holding potential set to the reversal potential for AMPA/NMDA-mediated current, 0 mV) and excitatory post-synaptic current (lower trace, holding potential set to the reversal potential for GABA-mediated current, −55 mV), recorded in a pyramidal cell in whole-cell voltage-clamp mode; scale bar: 40 pA, 20 ms. (e) Light stimulation specifically evoked inhibitory post-synaptic current (upper trace), no excitatory post-synaptic current was detected (lower trace); scale bar: 20 pA, 20 ms. Inset, both inhibitory and excitatory spontaneous synaptic events were detected using the same recording conditions; scale bar: 20 pA, 50 ms.
Figure 4.
In-vivo 2-photon imaging of GFP-labeled basket interneurons in Pv-cre mice.
(a) Low-magnification projection of a z-series starting at ∼20 µm below the pia surface to a depth of ∼120 µm, from a densely labeled area. The asterisks indicate the tops of two cell bodies. Numerous dendritic branches are indicated by blue arrows. (b–c) Higher magnification z-projections of regions highlighted in (a) at two different depths: 85–90 µm (b) and 65–75 µm (c) below the pia. Note the smooth, aspiny dendrites (blue arrows), and dense cluster of boutons of varying size (yellow arrowheads). (d) Projection of a z-series 60–170 µm below the pia from a sparsely labeled area, showing an isolated GFP-labeled basket interneuron. Dendrites (blue arrows) could be traced back to soma, and axonal boutons (yellow arrowheads) appear as a cloudy signal at this magnification. (e–f) Examples of axon morphology magnified from areas indicated by gray boxes in (d). (e) shows an axonal basket-like structure (asterisk), and (f) shows a well-isolated axon branch (yellow arrow) with distinct boutons (yellow arrowheads). (g–h) dendrite structures from areas indicated in (d). Dendrites of the cell were largely aspiny (g), though occasionally small protrusions were visible on some dendritic branches (h). Scale bars a,d: 20 µm; b–c, e–h: 5 µm.
Figure 5.
Chronic 2-photon imaging of structural dynamics of basket interneurons in-vivo.
(a) Example images of the morphology of an axon branch and boutons in which both were stable over the course of two days. (b) One-week imaging interval in which the axon branch and boutons were largely stable (yellow arrowheads), additions (blue arrowheads) and subtractions (red arrowheads) on the axon shaft were observed. Note, two far right boutons are not shown for day 7. (c) Proportion of axon bouton additions and subtractions (total number of boutons examined = 183, from three animals). (d) Example in which newly formed boutons persistented for at least two days (blue arrowheads). Scale bars: 5 µm.