PMC

Specific labeling of PV+ inhibitory interneurons in visual cortex of PV-Cre mice. (A) The viral construct contained a floxed-STOP codon followed by RFP under control of the CMV promoter (). (B, C) Immunohistochemical verification of RFP expression. Sections containing virally-infected cells were immunostained for either (B) PV, or (C) GABA. Virtually all RFP+ cells were both PV+ and GABA+. Inset in (B) and images in (C) were taken from the depth at which in vivo recordings were performed (~130–300µm). Scalebars: (B) 100µm, inset: 50µm, (C) 25µm.

The spatial receptive field sizes of the RFP+ and RFP− populations overlap significantly when measured with in vivo two-photon calcium imaging.
(A) The raw calcium indicator fluorescence response to each presentation of a periodically drifting checkered bar in the elevation axis (light traces), overlaid with the best-fit Gaussian (dark traces) for two representative RFP+ (red) and RFP− (black) cells. (B) Population histograms of the receptive field size (half width at half height) in the elevation and azimuth axes. Arrowheads indicate the mean of each distribution.

Spatial frequency tuning is similar in the RFP+ and RFP− populations when measured with in vivo two-photon calcium imaging and loose-patch electrophysiological recordings. (A) Calcium imaging data. (Top) The best Difference of Gaussians fit to the dF/F responses for two representative cells (see for raw traces of the data). RFP+ cell: Preferred spatial frequency (PSF) = 0.04 cpd, bandwidth = 5.1 octaves. RFP− cell: PSF = 0.04 cpd, bandwidth = 2.7 octaves. Error bars indicate SEM. (Bottom) Population histograms of the preferred spatial frequency and spatial frequency tuning bandwidth are shown for the RFP+ and RFP− populations. The low-pass bin denotes cells with no low spatial frequency roll-off. Arrowheads denote the mean of each distribution. (B) Loose-patch recording data. (Top) The best Difference of Gaussians fit to the spike responses of representative RFP+ and RFP− cells. RFP+ cell: PSF = 0.007 cpd, bandwidth = 4.7 octaves, RFP− cell: PSF = 0.03 cpd, bandwidth = 2.6 octaves. (Bottom) Population histograms of the preferred spatial frequency and spatial frequency tuning bandwidth.

Two-photon guided loose-patch recordings of PV+ interneurons reveal sharp orientation tuning in a subset of PV+ neurons. (A) RFP+ cells (red) were targeted with a patch pipette containing Alexa 488 dye (green). After the visual responses of each neuron were characterized, the cell was filled to confirm its identity. (B) Bar graphs show the population mean peak:valley amplitudes, spike widths and mean repolarization rates for RFP+ cells (red) and RFP− cells (black). Error bars indicate SEM. The mean peak:valley amplitude and repolarization rate were significantly different between the two populations (asterisk). (C) The mean spike, normalized to the peak value, of all RFP+ (red) and RFP− (black) cells is plotted. (D) Examples of orientation tuning curves from three RFP+ and three RFP− cells. The preferred direction was set to 180 degrees for ease of comparison of the tuning among cells. The waveform properties of these cells (1,2,3 and a,b,c) are indicated in . (1) OSI = 1.0, tuning width = 15.8 deg, DSI = 0.8. (2) OSI = 1.0, tuning width = 16.7 deg, DSI = 0.9. (3) OSI = 0.7, tuning width = 30.1 deg, DSI = 0.6. (a) OSI = 0.8, tuning width = 15.0 deg, DSI = 0.7. (b) OSI = 0.8, tuning width = 16.2 deg, DSI = 0.7. (c) OSI = 0.6, tuning width = 10.0 deg, DSI = 0.6. Error bars indicate SEM. (E) Population histograms of the OSI, tuning width, and DSI of RFP+ (red) and RFP− (black) cells. Arrowheads indicate the population means.

In vivo two-photon calcium imaging of RFP+ and RFP− neurons reveals extensive overlap in the orientation tuning properties of PV+ interneurons and the unlabeled population. (A) Two weeks after viral infection, a craniotomy was made, and the calcium indicator OGB was injected into the infected site. The RFP alone, OGB alone, and merged images are shown. Arrowheads point to the same cells in each image. Scalebar = 10µm. (B) (Left) Calcium indicator responses of representative RFP+ cells (red traces) and RFP− cells (black traces) to episodically-presented oriented gratings at 20-degree intervals; each grating was drifted in a direction orthogonal to the grating orientation (gray shading: ON periods of stimulus presentation, white: OFF). Raw single-trial traces (thin lines) as well as the mean response trace (thick lines) are shown. (Right) Gaussian tuning curves were fitted to the calculated ΔF/F responses for each stimulus, as described in Methods. The peak response is set to 180 deg for ease of comparison. Top RFP+ cell: OSI = 0.23, tuning width = 32 deg, DSI = 0.3. Bottom RFP+ cell: OSI = 0.53, tuning width = 19 deg, DSI = 0.5. Top RFP− cell: OSI = 0.13, tuning width = 81 deg, DSI = 0.5. Bottom RFP− cell: OSI = 0.48, tuning width = 12 deg, DSI = 0.5. Error bars on tuning curves denote SEM. (C) Population histograms of the orientation and direction tuning properties of RFP+ (red) and RFP− (black) populations show the extensive overlap between the two cell populations in OSI, tuning width, and DSI. Arrowheads on histograms mark the mean of each population.