Prolonged disynaptic inhibition in the cortex mediated by slow, non-α7 nicotinic excitation of a specific subset of cortical interneurons

Abstract

Cholinergic activation of nicotinic receptors in the cortex plays a critical role in arousal, attention, and learning. Here we demonstrate that cholinergic axons from the basal forebrain of mice excite a specific subset of cortical interneurons via a remarkably slow, non-α7 nicotinic receptor-mediated conductance. In turn, these inhibitory cells generate a delayed and prolonged wave of disynaptic inhibition in neighboring cortical neurons, altering the spatiotemporal pattern of inhibition in cortical circuits.

Figure 1.

Nicotinic receptor-dependent inhibitory barrage in L2/3 pyramidal cells. A, Experimental setup: The BF of ChAT-Cre mice was injected with an AAV vector driving Cre-dependent ChR2-EYFP expression in ChAT-expressing cells (green somas and fibers). B1, EYFP (left) and ChAT immunostaining (middle) colocalize (right), indicating selective transduction of ChAT-expressing cells. Scale bar, 50 μm. B2, Whole-cell recording from a virally transduced TdTomato labeled cell in the BF of a ChAT-Cre/TdTomato mouse. Left, Action potentials evoked during a step current injection; Right, A train of brief photostimulations (3 ms flashes at 6.7 Hz) evoked a train of action potentials in the cell. C, Confocal image showing ChR2-EYFP-expressing axons in cortex. D, Top, Diagram illustrating disynaptic inhibition generated by cholinergic axons; Bottom, Brief photostimulation (blue bar) evokes a barrage of PSCs in a L2/3 pyramid over multiple trials. Recordings were performed in DNQX (5 μm) with a high chloride internal solution (130 mm). E, Bath application of gabazine (10 μm) abolished the photostimulation-induced barrage; *p = 0.048, paired t test. F, Bath application of the nicotinic receptor antagonists MLA (5 nm) and DHβE (500 nm) abolished the photostimulation-induced barrage; *p = 0.005, paired t test. G, Cumulative incidence of barrage latencies for 14 L2/3 pyramidal cells. Latencies were computed for each cell from an average of several trials. H, Cumulative incidence of barrage duration for same cells as in G.

Figure 2.

Responses to cholinergic fiber stimulation in four types of interneurons in layer 1 and layer 2/3. A, Characteristic firing pattern of a layer 1 (L1) interneuron in response to current injection (left) and a representative response of the same cell to photostimulation of cholinergic fibers (tick mark below trace; right). The dotted boundary indicates the region expanded in the inset (upper right). Inset scale bar applies to all insets below. Black lines in all insets represent photostimulation. B, Same as A, but for a L2/3 LS cell. C, Same as A, but for a L2/3 CB cell. D, Same as A, but for a L2/3 FS cell. Scale bars apply to traces above. E, Input resistance, time constant, AHP, and spike width are plotted for L1, L2/3 LS cells, L2/3 CB, and L2/3 FS cells; *p < 0.05, paired t test.

Figure 3.

Inhibitory barrage in layer 2/3 fast-spiking cells. A, Diagram illustrating disynaptic inhibition in FS interneurons generated by cholinergic axons. B, Action potentials evoked in response to current injection for an FS cell. C, Photostimulation evoked an IPSC barrage in this cell. D, The photostimulation-evoked IPSC barrage is abolished by DHβE (500 nm) together with MLA (5 nm).

Figure 4.

Cholinergic axons elicit a prolonged nicotinic excitation in cortical interneurons. A, Response of a L1 cell to brief (3 ms) photostimulation in control conditions (black) and in 5 nm MLA and 500 nm DHβE (red). Superimposed gray traces represent single trial responses. B, Voltage-clamp recordings from two L1 interneurons reveals two nicotinic receptor-mediated components. Application of 500 nm DHβE, a non-α7 nicotinic receptor antagonist, selectively blocks the slow EPSC (left, control in black). In contrast, the selective α7 receptor antagonist MLA (5 nm) abolishes the fast component (right, control in black). C, Photostimulation-evoked bursts of action potentials in a L1 cell and a L2/3 CB cell. D, Spike latency histogram for all spiking L1 and L2/3 CB cells. Counts for each cell type are stacked. E, Charge transferred by the pharmacologically isolated fast and slow EPSCs. Traces above the bar graph depict the population-averaged waveforms of the fast (n = 6) and slow (n = 4) EPSCs; *p = 0.005, t test. F, The time course of the averaged IPSC barrage recorded in pyramidal cells (n = 4 cells, green) is superimposed on the averaged fast EPSC (isolated with DHβE; n = 6 cells, light gray) and slow EPSC (isolated with MLA; n = 4 cells, dark gray) for comparison. G, Application of DHβE spares the fast component of the nicotinic EPSC but abolishes the slow nicotinic EPSC and the IPSC barrage (n = 3).