The Basal Forebrain and Motor Cortex Provide Convergent yet Distinct Movement-Related Inputs to the Auditory Cortex

Abstract

Cholinergic inputs to the auditory cortex from the basal forebrain (BF) are important to auditory processing and plasticity, but little is known about the organization of these synapses onto different auditory cortical neuron types, how they influence auditory responsiveness, and their activity patterns during various behaviors. Using intersectional tracing, optogenetic circuit mapping, and in vivo calcium imaging, we found that cholinergic axons arising from the caudal BF target major excitatory and inhibitory auditory cortical cell types, rapidly modulate auditory cortical tuning, and display fast movement-related activity. Furthermore, the BF and the motor cortex-another source of movement-related activity-provide convergent input onto some of the same auditory cortical neurons. Cholinergic and motor cortical afferents to the auditory cortex display distinct activity patterns and presynaptic partners, indicating that the auditory cortex integrates bottom-up cholinergic signals related to ongoing movements and arousal with top-down information concerning impending movements and motor planning.

Figure 1. Cholinergic SIn Neurons Target the Major Cell Types of ACtx

(A-C) Overview of the rabies-based monosynaptic, presynaptic tracing strategy. (A) On day 0, AAV-FLEX-RG and AAV-FLEX-TVA.mCherry were injected into ACtx of a Cre driver mouse, in this case PV-Cre. (B) On day 14, EnVA-R∆G.GFP was injected into ACtx, which labeled neurons presynaptic to PV cells expressing TVA and RG (C). (D) GFP and mCherry labeling in ACtx. (E) Inset from (D) showing GFP⁺, mCherry⁺, and double-labeled neurons. (F) GFP⁺ neurons (presynaptic to PV neurons in ACtx) in MGB. (G) GFP⁺ neurons in contralateral ACtx. (H) GFP⁺ neurons in SIn. The lower left inset diagrams the target of SIn neurons for this experiment. The upper right inset indicates the location of SIn_ACtx labeling on a coronal brain slice (from Paxinos & Franklin). (I-K) Top panels indicate the location and target cell type of the GFP⁺ labeling in lower panels. (L) The coronal brain coordinates for SIn_ACtx neurons presynaptic to four cell types of ACtx, relative to a common midline landmark and normalized by brain slice size (n = 2 mice for each plot). The color code indicates rostral-caudal location of labeled neurons. (M) Locations of SIn neurons targeting different ACtx cell types superimposed on a generalized coronal brain slice (n = 8 mice total). (N) ChAT immunolabeling in GFP+ SIn neurons targeting PV⁺ ACtx neurons. Double-labeled SIn_ACtx neurons (SIn_ACh neurons are indicated with arrowheads). Panels to the right show GFP⁺ neurons (top), ChAT⁺ neurons (middle), and overlaid images (bottom). (O) Percent ChAT⁺GFP⁺ SIn neurons for each target ACtx cell type (CaMKII, n = 3; PV, n = 2; VIP, n = 3; SST, n = 3). (n.s. p > 0.05). Values are mean ± SEM.

Figure 2. SIn_ACh Neurons evoke excitatory and inhibitory currents in ACtx neurons

(A) Schematic of the experimental strategy. AAV-FLEX-ChR2-GFP was injected into SIn of ChAT-Cre mice. Whole-cell voltage clamp recordings were made from acute coronal ACtx brain slices while photostimulating ChR2-expressing SIn_ACh axons. (B-D) Immunoreactive ChR2-expressing SIn_ACh axons (green) around a pyramidal neuron labeled following whole-cell recording (red). (B) Axons surrounding the soma. (C) Axons surrounding the proximal trunk dendrite. (D) Axons surrounding a segment of apical dendrite in layer 1 of cortex. (E-H) Average (4 trials each) excitatory and inhibitory currents evoked in a pyramidal neuron through stimulation of SIn_ACh axons in normal ACSF (nACSF, E), ACSF containing NBQX, AP5, & atropine (F), NBQX, AP5, atropine, and GBZ (G), or NBQX, AP5, atropine, GBZ, and mecamylamine (H). (I) Peak excitatory and inhibitory currents evoked through stimulation of SIn_ACh axons in various pharmacological conditions for a population of neurons. (J) Onset times for excitatory and inhibitory currents (n = 12 neurons from 7 mice). The inset depicts at higher magnification a neuron's response to SIn_ACh stimulation at holding potentials of −70mV (grey) and 0mV (black). (K) Grand average of excitatory currents evoked through stimulation of SIn_ACh axons after applying NBQX, AP5, atropine, and GBZ. Lighter shade traces represent SEM (n = 8 neurons. Values are mean ± SEM.

Figure 3. SIn_ACh Neurons Depolarize ACtx Neurons

(A) Schematic of the experimental strategy. AAV-FLEX-ChR2-GFP was injected into SIn of ChAT-Cre mice. Sharp intracellular current clamp recordings were made from mice while photostimulating ChR2-expressing SIn_ACh axons and playing sounds. The insets show ChR2-expressing SIn_ACh axons (green) around a pyramidal neuron labeled following intracellular recording (red). (B) Average response (20 trials, blue trace) to photostimulating SIn_ACh axons (blue shaded bar) for an example neuron. The black trace shows average response to blank stimulation trials. (C) Average voltage area following stimulation of SIn_ACh axons or blank trials for the example neuron in (B). (D) Average spike count following stimulation of SIn_ACh axons or blank trials for the example neuron in (B). (E) Grand average intracellular response to stimulation of SIn_ACh axons (n = 10 neurons from 3 mice). Shaded bounds indicate SEM. (F) Population depiction of evoked voltage area. The blue data points indicate the population mean and SEM. (G) Average response (40 trials) to tone playback alone (black trace) or tone playback with simultaneous photostimulation of SIn_ACh axons (blue trace) for the example neuron. (H) Mean voltage area in response to tone playback alone (black) or tone playback with stimulation of SIn_ACh axons (blue) for the example neuron. (I) Mean spiking response to tone playback alone (black) or tone playback with stimulation of SIn_ACh axons (blue) for the example neuron. (J) Grand average intracellular response to tone playback alone (black) or tone playback with stimulation of SIn_ACh axons (blue). Lighter shades depict SEM. (K) Population depiction of voltage area evoked to tone presentation alone or with stimulation of SIn_ACh axons. The blue data points indicate the population mean and SEM. Values are mean ± SEM.

Figure 4. SIn_ACh Neurons Facilitate Weak and Suppress Strong Auditory Responses

(A) Schematic of the experimental strategy. AAV-FLEX-ChR2-GFP was injected into SIn of ChAT-Cre mice. Multielectrode extracellular recordings were made from ACtx while photostimulating ChR2-expressing SIn_ACh axons and playing sounds. (B) Raster plots for an isolated single unit in response to tone playback (2 kHz, blue or 4 kHz, orange) alone (bottom half), or with photostimulation of SIn_ACh axons (top half). The blue shaded box indicates the light stimulation period. (C) Peristimulus time histograms of response strength (RS) to tone stimulation alone (solid blue/orange traces), or tone + light stimulation (dashed blue/orange traces). (D) Tuning curve for the example unit without (solid trace) or with (dashed trace) stimulation of SIn_ACh axons. (E) Tone RS without light plotted against tone RS with light, for a single experiment. All significantly driven tone responses for each unit are included as individual data points. On average, tone responses are facilitated with light (p = 0.0058). The lighter blue shading indicates the functional prediction bounds for the linear regression. (F) Population tuning curve without (black) and with (blue) light stimulation (data are from 5 mice). (G) Population tone RS plotted against population tone + light RS for each group of responses (± 0, 1, 2, 3 octaves from peak response). The blue line is the linear fit. Values are mean ± SEM.

Figure 5. SIn_ACh Axons in ACtx are Active During Movements

(A) Schematic of the experimental strategy. AAV-FLEX-GCaMP6s was injected into SIn of ChAT-Cre mice. (B) Confocal micrograph of GCaMP-labeled SIn neurons. Neurons expressing GCaMP expressed ChAT (red). (C) Confocal micrograph of GCaMP-expressing SIn_ACh axons in ACtx. (D) Outline of experimental design for 2 photon imaging and tracking movements. ROIs for monitoring body movements are indicated with dashed boxes. (E) Average fluorescence of a GCaMP-labeled axon, imaged in vivo. The region outlined with a green dashed line indicates the ROI for monitoring changes in fluorescence over time. (F) Pseudocolored axonal fluorescence during rest. (G) Pseudocolored axonal fluorescence during a body movement. (H) ∆F/F from a single ROI (blue trace) during movements and rest (red trace). The dashed line indicates the threshold for identifying movements. (I) ∆F/F during paw movements (n = 12 axons, 2 mice). (J) ∆F/F during mouth movements (n = 12 axons, 2 mice). (K) ∆F/F during all body movements (n = 14 axons, 3 mice). (**p < 0.01, *** p < 0.001). Values are mean ± SEM.

Figure 6. Movement-Related SIn_ACh Activity Precedes Pupil Microdilations

(A) Outline of experimental design for 2 photon imaging, tracking movements, and monitoring pupil size. (B) Changes in pupil size across time. Representative images from dilated, constricted, and microdilation periods are shown above. The dashed white circle corresponds to the size of the constricted pupil, while the orange circles illustrate the relative dilations. (C) ∆F/F from a single axon (blue) and pupil size (purple) during an epoch of small body movements (red). The lighter purple trace shows the unfiltered pupil size. (D) Cross correlation between ∆F/F of 9 ROIs and pupil microdilations. The lag between ∆F/F and pupil is indicated by the dashed orange line relative to time zero. The inset shows GCaMP labeling in SIn_ACh axons, and the ROIs selected for analysis. (E) Cross correlation between ∆F/F of the same ROIs from (D) and movement quantification. (F-G) Average and SEM of ∆F/F (blue trace, n = 14 imaging planes, 1 axon from each; 4 mice) and pupil area (purple trace, n = 5 imaging sessions, 2 mice) plotted with respect to movement onset (F) and movement offset (G). Values are mean ± SEM (shading), except for the horizontal error bars in (F), which represent SD.

Figure 7. SIn_ACh and M2_ACtx Synapses Converge on Single Auditory Cortical Neurons

(A) Schematic of the experimental strategy. AAV-FLEX-GFP was injected into SIn of ChAT-Cre mice. AAV-tdTomato was injected into the M2 of the same mice. (B) Confocal micrograph showing GFP labeling at the injection side. Blue is DAPI. The inset shows the injection site, immunostained for ChAT (red). (C) tdTomato labeling at the M2 injection site. (D) Labeling from SIn_ACh (green) and M2_ACtx (red) axons in ACtx. The inset shows the fluorescence intensity of SIn_ACh and M2_ACtx labeling across layers (averaged measurements from several brain sections). (E) Higher magnification Z stack showing SIn_ACh and M2_ACtx axons near a putative cell body (asterisk). (F) Higher magnification image from (E). The Z stack was interpolated and rotated slightly. Nicotinic ACh receptors are labeled with ɑ-bungarotoxin (greyscale). (G) Another Z stack showing SIn_ACh and M2_ACtx axons near a cell body from a different mouse. DNA is labeled with DAPI. (H) Higher magnification Z stack from (G). (I) Schematic of the experimental strategy. AAV-FLEX-ChR2-GFP was injected into SIn of ChAT-Cre mice. AAVChR2-GFP was injected into the M2 of the same mice. Whole-cell voltage clamp recordings were made from acute coronal ACtx brain slices. (J) Confocal Z stack showing a pyramidal neuron labeled following whole-cell recording surrounded by ChR2-expressing axons. (K) Average traces of excitatory currents (5 neurons) in response to photostimulation of putative SIn_ACh and M2_ACtx axons in normal ACSF (blue) and following application of blockers (orange; the shading indicates SEM). Nicotinic transmission is preserved after blocking glutamatergic M2_ACtx currents. (L) Onset (blue) and offset (magenta) time constants of excitatory currents from (J).

Figure 8. Synaptic Inputs to SIn_ACtx Neurons

(A) Schematic of the experimental strategy. On day 0, AAV-FLEX-RG and AAV-FLEX-TVA.mCherry were injected into SIn of wild type mice. Additionally, CAV-Cre was injected into ipsilateral ACtx. On Day 14, EnVA-R∆G.GFP is injected into SIn, which labeled neurons presynaptic to SIn_ACtx cells expressing TVA and RG. (B) GFP and TVA.mCherry labeling in SIn. (C) GFP-labeled SIn neurons. (D) TVA.mCherry-labeled SIn_ACtx neurons. (E) Overlaid images from (C) and (D). Neurons expressing both GFP and TVA.mCherry are starter cells. Neurons expressing GFP, but not mCherry, are putatively presynaptic to SIn_ACtx neurons. (F) A subset of SIn neurons putatively presynaptic to SIn_ACtx neurons stained positive for NPY (white arrowheads). The panels to the right show GFP (top), NPY (middle), and TVA.mCherry (bottom) labeling. The white arrowheads indicate a putative presynaptic (GFP⁺/TVA.mCherry⁻) neuron expressing NPY. The open yellow arrowhead indicates a starter cell (GFP⁺/TVA.mCherry⁺) not expressing NPY. (G-O) Neurons presynaptic to SIn neurons in cuneiform nucleus (G), peripeduncular nucleus and suprageniculate nucleus (H), periaqueductal gray (I), locus coeruleus (J), raphe (K), hypothalamus (N), and frontal cortex (O).