Synapse-associated protein 97 regulates the membrane properties of fast-spiking parvalbumin interneurons in the visual cortex

Abstract

Fast-spiking parvalbumin (PV)-positive interneurons in layers 2/3 of the visual cortex regulate gain control and tuning of visual processing. Synapse-associated protein 97 (SAP97) belongs to a family of proteins that have been implicated in regulating glutamatergic synaptic transmission at pyramidal-to-pyramidal connections in the nervous system. For PV interneurons in mouse visual cortex, the expression of SAP97 is developmentally regulated, being expressed in almost all juvenile but only a fraction, ~40%, of adult PV interneurons. Using whole-cell patch-clamping, single-cell RT-PCR to assay endogenous expression of SAP97 and exogenous expression of SAP97, we investigated the functional significance of SAP97 in PV interneurons in layers 2/3 of the visual cortex. PV interneurons expressing SAP97, either endogenously or via exogenous expression, showed distinct membrane properties from those not expressing SAP97. This included an overall decrease in membrane excitability, as indexed by a decrease in membrane resistance and an increase in the stimulus threshold for the first action potential firing. Additionally, SAP97-expressing PV interneurons fired action potentials more frequently and, at moderate stimulus intensities, showed irregular or stuttering firing patterns. Furthermore, SAP97-expressing PV interneurons showed increased glutamatergic input and more extensive dendritic branching when compared with non-expressing PV interneurons. These differences in membrane and synaptic properties would significantly alter how PV interneurons expressing SAP97 compared with those not expressing SAP97 would function in local networks. Thus, our results indicate that the scaffolding protein SAP97 is a critical molecular factor regulating the input-output relationships of cortical PV interneurons.

Figure 1.

In PVdT mice, tdTomato colocalizes with PV. Confocal images of the visual cortex from juvenile (A₁–A₄) or adult (B₁–B₄, C₁–C₄) PVdT mice. Tissue sections were labeled with either rabbit anti-PV (A₁, B₁) or rat anti-SOM (C₁) and visualized for either PV (green; A₁, B₁) or SOM (green; C₁) and tdTomato (red; A₂, B₂, C₂) signals. Merged images are shown in A₃,A₄, B₃,B_4, and C₃,C₄. The images are at either 10× (A₁–A₃, B₁–B₃, C₁–C₃) or 63× (A₄, B₄, C₄). Scale bars: 10× images, 100 μm; 63× images, 50 μm. The higher-magnified images (A₄, B₄, C₄) show a smaller region in layers 2/3 of the merged images in A₃, B₃, and C₃. The specificity of the PV and SOM antibodies has been verified (Akgul and Wollmuth, 2010). Merged images show either colocalization of PV and tdTomato (A₃,A₄ and B₃,B₄) or lack of overlap between SOM and tdTomato (C₃,C₄).

Figure 2.

SAP97⁺ PV interneurons display distinct membrane properties from those not expressing SAP97. Analysis of membrane properties in juvenile PV interneurons either expressing (first column, Juvenile/97+) or not expressing (second column, Juvenile/97−) SAP97 or adult PV interneurons either expressing (third column, Adult/97+) or not expressing (last column, Adult/97−) SAP97. Each column represents the results from the same interneuron. A₁–A₄, Gene expression profiles of individual interneurons. GAD67 and GAD65 are GABAergic interneuron markers, and PV is PV interneuron marker. GAD67 and GAD65 are used as positive controls. VGluT1 and no template (no temp) lanes are negative controls. B₁–B₄, Voltage traces (V_m) of individual interneurons firing at ∼30 Hz. The depolarizing currents (I_m) to elicit 30 Hz firing are 790, 310, 340, and 200 pA, respectively. C₁–C₄, Voltage traces (V_m) for the hyperpolarized membrane potential and the first AP in response to rheobase current are shown along with the injected current traces. Rheobase currents are 720, 190, 200, and 150 pA, respectively. D₁–D₄, The rheobase APs are expanded (10 ms total time) to highlight differences in AP shape.

Figure 3.

SAP97⁺ and SAP97⁻ PV interneurons receive different excitatory input. A₁–A₄, Representative current traces (10 s) from individual juvenile or adult PV interneurons either expressing (97+) or not expressing (97−) SAP97 recorded in whole-cell voltage clamp (V_hold = −70 mV) in the presence of Mg²⁺, TTX, and picrotoxin. B₁–B₄, Segments of the same current traces expanded to show 100 ms of recording. C₁–C₄, Cumulative histograms of mEPSC frequency (C₁, C₃) and amplitude (C₂, C₄). Continuous lines indicate SAP97+ (juvenile, n = 8; adult, n = 6), and dashed lines indicate SAP97⁻ (juvenile, n = 5; adult, n = 13) interneurons. mEPSC frequency distributions in both juvenile and adult SAP97⁺ interneurons are significantly different from those in SAP97⁻ interneurons (p < 0.01, KS test). mEPSC amplitude distributions are significantly different between SAP97⁺ and SAP97⁻ interneurons only in adult (p < 0.01, KS test). D₁–D₄, Bar graphs of mEPSC frequency (D₁), amplitude (D₂), half-width (D₃), and decay (D₄). Values shown are mean ± SEM (for details, see Materials and Methods). *p < 0.05, **p < 0.01, Student's t test. 97-, SAP97⁻; 97+, SAP97⁺; J, juvenile; A, adult. Average access resistance and number of events for each groups are as follows: J/97+, 5.6 ± 0.3 MΩ and 1423 ± 145 events; J/97−, 5.4 ± 0.4 MΩ and 1360 ± 91 events; A/97+, 4.7 ± 0.4 MΩ and 1412 ± 124 events; and A/97−, 4.7 ± 0.4 MΩ and 1226 ± 67 events.

Figure 4.

Exogenous expression of SAP97egfp in PV interneurons. Confocal images of the visual cortex from adult (A₁–A₄) PVdT mice. FLEX–rev-a–SAP97–EGFP_AAV was stereotaxically injected into the visual cortex. Tissue sections were visualized for either EGFP (green; A₁, A₄) or tdTomato (red; A₂) signals. Merged image is shown in A₃. The images are at either 10× (A₁–A₃) or 63× (A₄). Scale bars: 10× images, 100 μm; 63× images, 50 μm. The higher-magnified image (A₄) shows a smaller region in layers 2/3 of A₁. Merged image shows colocalization of EGFP and tdTomato, and the magnified image shows somatodendritic localization of punctate EGFP signal.

Figure 5.

Membrane properties of adult PV interneurons exogenously expressing SAP97egfp. A, Gene expression profile of an individual adult PV interneuron exogenous expressing SAP97egfp (Adult/97egfp). PV and SAP97 primers target PV and SAP97 open reading frame, and 3′UTR primers target the 3′UTR region of SAP97 to detect endogenous SAP97. No template (no temp) is negative control. 3′UTR primers were also used on cDNA preparation of isolated total cortical RNA as a positive control. B, Voltage trace (V_m) of an individual interneuron firing at ∼30 Hz. The depolarizing current (I_m) to elicit 30 Hz firing is 420 pA. Bar graphs show Max/Min ISI and Min ISI for SAP97⁺ (97+), SAP97⁻ (97−), and SAP97egfp (97egfp) exogenously expressing adult PV interneurons. C, The membrane potential in response to hyperpolarizing current (−40 pA) and the first AP firing in response to rheobase current (280 pA). Bar graphs show R_m and rheobase. D, The rheobase AP is expanded (10 ms total time). Bar graphs show AP half-width (HW) and AHP area. E, Bar graphs show Min ISI (E₁), Max ISI (E₂), and Max/Min ISI (E₃) for SAP97⁺ (gray), SAP97⁻ (white), and SAP97egfp (black) exogenously expressing adult PV interneurons at increasing stimulus steps above rheobase (>10 to 50 pA). Values shown in bar graphs are mean ± SEM. All values are compared with the SAP97⁻ group. *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test.

Figure 6.

PV interneurons exogenously expressing SAP97egfp show higher levels of excitatory activity. Analysis of mEPSCs in adult PV interneurons exogenously expressing SAP97egfp compared with SAP97⁻ and SAP97⁺ adult interneurons. A, Representative current trace (10 s) from an individual interneuron recorded in whole-cell voltage clamp (V_hold = −70 mV) in the presence of Mg²⁺, TTX, and picrotoxin. B, Segments of the same current trace are expanded to show 100 ms recording. C₁, C₂, Cumulative histograms of mEPSC frequency (C₁) and amplitude (C₂). Dashed line indicates SAP97⁻ (n = 13), dotted line indicates SAP97⁺ (n = 6), and solid line indicates SAP97egfp (n = 5). Both frequency and amplitude distributions for SAP97⁺ and SAP97egfp interneurons are significantly different from those for SAP97⁻ (p < 0.01, KS test). D₁–D₄, Bar graphs of mEPSC frequency (D₁), amplitude (D₂), half-width (D₃), and decay (D₄). Values shown in bar graphs are mean ± SEM. *p < 0.05, **p < 0.01. 97−, SAP97⁻; 97+, SAP97⁺; 97egfp, SAP97egfp. Average access resistance and number of events for SAP97egfp: 5.1 ± 0.2 MΩ and 1560 ± 180 events.

Figure 7.

SAP97⁺ PV interneurons show more extensive dendritic branching. A, B, Representative micrographs (A₁, B₁) and Imaris traces (A₂, B₂) of biocytin-filled SAP97⁻ and SAP97egfp PV interneurons. Scale bars, 50 μm. C, Sholl analysis in SAP97⁻ (n = 4), SAP97⁺ (n = 4), and SAP97egfp (n = 3) interneurons. *p < 0.05, SAP97⁺ or SAP97egfp significantly different from SAP97⁻.