Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo

Abstract

It is becoming increasingly clear that single cortical neurons encode complex and behaviorally relevant signals, but efficient means to study gene functions in small networks and single neurons in vivo are still lacking. Here, we establish a method for genetic manipulation and subsequent phenotypic analysis of individual cortical neurons in vivo. First, lentiviral vectors are used for neuron-specific gene delivery from alpha-calcium/calmodulin-dependent protein kinase II or Synapsin I promoters, optionally in combination with gene knockdown by means of U6 promoter-driven expression of short-interfering RNAs. Second, the phenotypic analysis at the level of single cortical cells is carried out by using two-photon microscopy-based techniques: high-resolution two-photon time-lapse imaging is used to monitor structural dynamics of dendritic spines and axonal projections, whereas cellular response properties are analyzed electrophysiologically by two-photon microscopy directed whole-cell recordings. This approach is ideally suited for analysis of gene functions in individual neurons in the intact brain.

Fig. 1.

Lentivirus-based EGFP expression in cortical neurons in vivo: P21–P28 infection. (a) Examples of lentivirus infected layer 2/3 neurons in the somatosensory cortex. The internal promoter types and corresponding vector names are indicated at the top and bottom of each image, respectively. The images are maximum-intensity projections of five z sections separated by 1.0 μm, collected by confocal microscopy from the outer region of the injection sites (within 100 μm from the periphery of an injection area of a 500- to 600-μm diameter) in brain sections. [Scale bar, 10 μm (Left Upper) is valid for all images.] (b) Quantification of EGFP expression based on fluorescent signal within nuclei of individual infected neurons (n = number of cells analyzed). The promoter types and corresponding vector names are indicated above and below the bar graphs, respectively. The values (percent mean ± SEM) are normalized to FUGW expression: FUGW = 100 ± 6.9, n = 69; FCbAGW = 62.7 ±1.8, n = 13; FThGW = 74.8 ± 3.9, n = 19; FCK(0.4)GW = 155.1 ± 7.9, n = 20; FCK(1.3)GW = 165.7 ± 13.5, n = 41; FCK(2.4)GW = 41.1 ± 1.0, n = 30; FSy(0.5)GW = 142.5 ± 4.7, n = 20; FSy(1.1)GW = 119.4 ± 5.2, n = 20.

Fig. 2.

Lentivirus-based EGFP expression in cortical neurons in vivo: P11 to P15/18 infection. (a) Examples of infected layer 2/3 neurons in the somatosensory cortex, as in Fig. 1a. The infection period is indicated on the left. (b) Quantification of EGFP expression normalized to FCK(1.3)GW expression, as in Fig. 1. The infection period is indicated above the bar graphs. P11–P15: FCK(1.3)GW = 100 ± 13.6, n = 10; FSy(1.1)GW = 197.9 ± 7.1, n = 10; P11-P18: FCK(1.3)GW = 100 ± 10.3, n = 8; FSy(1.1)GW = 170.4 ± 6.9, n = 13.

Fig. 3.

In vivo expression pattern of cortical layer 2/3 infected neurons. Each image is a maximum-intensity side projection from an overview stack of fluorescence images recorded by using in vivo two-photon microscopy. Note the different depth scaling (indicated on the left of each image) and the different scale bars. (a) FCK(1.3)GW-infected neurons in P28 rat after 7 days of expression. (b) FSy(1.1)GW-infected neurons in P48 mouse after 8 days of expression.

Fig. 4.

Two-photon time-lapse imaging of dendritic spines and axonal projections in the mouse cortex. Acquisition times are indicated on the sides of each image. Each image is a maximum-intensity projection of image stacks (z step, 1 μm) collected in cortical layer 1 in vivo (infected neurons were located in layer 2/3). Dashed vertical lines serve as a guide for comparison of structures over time. (a) Imaging of dendritic spines caused no photobleaching after 30 min at a 1-min sampling interval, with ▵F(t = 30 min) ≈0.97·▵F(t = 0min). (b) Imaging of axons in a layer 1 region adjacent to the injection site for >5 h at a 5-min sampling interval, with ▵F(t = 300 min) ≈0.66·▵F(t = 0 min). Some axonal projection endings, an example indicated by an arrow, showed directed outgrowth for several micrometers on a time scale of several hours in young animals (P15–P17 mice). Note the different scale bars in a and b.

Fig. 5.

Physiological analysis of infected neurons by TPTP recording. (a) An example of an infected in vivo-patched neuron. The EGFP image shows FCK(1.3)GW-infected layer 2/3 cells, at a depth of 182 μm below pia; The Alexa 594 image shows a patch pipette in a whole-cell configuration on the infected cell shown in the center on the left. (b) Horizontal projection of dendritic arbors relative to position of layer 4 barrels of the patched infected neuron shown in a. The cell was reconstructed from tangential sections. (c) Spontaneous activity and firing pattern of FCK(1.3)GW-infected layer 2/3 neuron shown in a. Top trace shows typical two-state membrane fluctuations (up and down states) observed in cortical neurons in anesthetized animals; bottom trace shows spiking in the same cell, elicited with DC current injection. In total, nine regular spiking FCK(1.3)GW-infected neurons were recorded, with average resting membrane potential, Vrest (mV) = 69.3 ± 2.0; steady-state input resistance, Rin (MΩ) = 65.3 ± 9.3; depolarization required for AP initiation (mV) = 30.7 ± 1.7.(d) Examples of whisker deflection-evoked responses in the same cell. Deflection of the D1, D2, and D3 whiskers, as illustrated in the schema on the left, evoked the corresponding responses shown on the right. The time course of the whisker deflection is shown at the bottom.