Precisely Timed Nicotinic Activation Drives SST Inhibition in Neocortical Circuits

Abstract

Sleep, waking, locomotion, and attention are associated with cell-type-specific changes in neocortical activity. The effect of brain state on circuit output requires understanding of how neuromodulators influence specific neuronal classes and their synapses, with normal patterns of neuromodulator release from endogenous sources. We investigated the state-dependent modulation of a ubiquitous feedforward inhibitory motif in mouse sensory cortex, local pyramidal (Pyr) inputs onto somatostatin (SST)-expressing interneurons. Paired whole-cell recordings in acute brain slices and in vivo showed that Pyr-to-SST synapses are remarkably weak, with failure rates approaching 80%. Pharmacological screening revealed that cholinergic agonists uniquely enhance synaptic efficacy. Brief, optogenetically gated acetylcholine release dramatically enhanced Pyr-to-SST input, via nicotinic receptors and presynaptic PKA signaling. Importantly, endogenous acetylcholine release preferentially activated nicotinic, not muscarinic, receptors, thus differentiating drug effects from endogenous neurotransmission. Brain state- and synapse-specific unmasking of synapses may be a powerful way to functionally rewire cortical circuits dependent on behavioral demands.

Keywords: attention; barrel cortex; endogenous neuromodulators; failure rate; glutamatergic; nicotinic; presynaptic release; rewiring; somatostatin; sparse coding.

Figure 1.. Local excitatory synaptic transmission onto layer 2/3 SST neurons is suppressed by network activity.

(A) The averaged trace of 10 response trials for a synaptic connection from a L2/3 pyramidal neuron to an SST interneuron (Pyr-SST) under normal (right) and elevated Ca²⁺ recording conditions. Ten presynaptic spikes (dashed vertical lines) at 20 Hz were delivered. Heatmaps at bottom show response amplitude for 10 trials (columns) with 10 spike pulses each (rows), using a linear scale where red scaled to the maximum amplitude. (B) Individual SST (red) response to a naturalistic spike train in pyramidal neuron (black), below 4 subsequent examples at higher time scale. Gray bars indicate individual spikes. Heatmap (right) shows response amplitude for 25, naturalistic spike trials, using a linear scale where red is maximum amplitude. (C) Connection probability in both conditions in slice and in vivo. (D) Mean EPSP amplitude in response to the first spike in the train, for 1 mM and 2.5 mM Ca²⁺ conditions in acute brain slice, and in vivo. EPSP amplitude is significantly different only between 1 and 2.5 mM Ca²⁺. (E) Mean failure rate after the first spike, for all conditions. For all, open circles represent individual cell measurements and filled circles represent all-cell mean± SEM, respectively (D,E).

Figure 2.. Screening for modulatory systems regulating Pyr-SST synaptic transmission.

(A) The -fold change of the first EPSP amplitude in a baseline condition and the following bath-applied agents: forskolin (cell-permeable activator of adenylyl cyclase ); carbachol (a broad-spectrum cholinergic agonist), adenosine; AM-251 (endocannabinoid inhibitors); depolarization-induced suppression of excitation (DSE); DHPG (a metabotropic glutamate receptor antagonist); serotonin; CY 208–243 (D1 receptor agonist) and norepinephrine. (B) The -fold change of the mean failure rate for the same conditions as in (A). Open circles represent individual cell values; bars represent all-cell mean± SEM.

Figure 3.. Nicotinic receptors enhance EPSP efficacy onto Pyr-SST connections.

(A) The averaged trace of 10 response trials for a Pyr to SST connection under baseline conditions (left) and in carbachol. Ten presynaptic spikes (dashed vertical lines) at 20 Hz were delivered. Heatmaps at bottom show response amplitude as in Figure 1. (B) Mean EPSP amplitude in response to the first spike in the train, for baseline and in carbachol conditions. (C) Mean failure rate after the first spike, for both conditions. (D) and (E) The same as in (B) and (C) but with selective nicotinic receptor activation (carbachol and atropine, a muscarinic receptor antagonist). (F) and (G) The same as in (B) and (C) but with selective muscarinic receptor activation (carbachol and mecamylamine, a nicotinic receptor antagonist). For all, open circles represent individual cell measurements and filled circles represent all-cell mean± SEM, respectively (B-G).

Figure 4.. Nicotinic receptors enhance EPSP efficacy at Pyr-SST connections.

(A) Schematic of the stimulation protocol. 1-single blue light (10 ms) was delivered 200 ms prior to the presynaptic spike train. (B) The averaged trace of EPSP under baseline/light OFF and light ON conditions. Heatmaps at bottom show response amplitude for both conditions as described in previous figures. (C) Mean EPSP amplitude in response to the first spike in the train, for both conditions. (D) Mean failure rate after the first spike, for both conditions. (E) and (F) The same as (C) and (D) but in the presence of the nicotinic receptors antagonist (mecamylamine). For all, open circles represent individual cell measurements and filled circles represent all-cell mean± SEM, respectively (C-F). See also Figure S1 and S2.

Figure 5.. SST neuron spontaneous firing with pharmacological and optogenetic ACh receptor activation.

(A) Firing frequency in baseline and in the presence of carbachol, carbachol and atropine, carbachol and mecamylamine. (B) The example traces showing the spike (top) and depolarization (bottom) of SST neurons in response to 10 ms-single light stimulation. (C) Mean fold change in spontaneous firing frequency for different conditions: carbachol, carbachol and atropine, carbachol and mecamylamine, light ON. The bar graphs represent mean±SEM.

Figure 6.. Nicotinic receptors enhance EPSP efficacy at Pyr-SST connections in vivo.

(A) Schematic of the experimental procedure. P10 SST-IRES-Cre-Ai9 pups were injected in nucleus basalis (Bregma: 0.02 mm, lat: 1.3 mm, depth: 4.5 mm). After an incubation period of 2–4 weeks mice were anesthetized and electrophysiological recordings were performed in somatosensory cortex. (B) Schematic of the recording setup. (C) In vivo 2-photon image of a pyramidal neuron (green cell soma) connected to a SST interneuron (yellow cell soma). White dashed lines show recording electrodes outlines. (D) Example recording of a Pyr (black trace) connected to SST (red trace). Bottom trace shows injected current into Pyr to drive spiking. (E) Zoom of the grey rectangle in (D). In this example two spikes were evoked and only the second one led to an EPSP in the SST interneuron (red trace). (F) Top: schematic of the experimental protocol: 1 blue light pulse (10 ms) was delivered on the brain surface (5-15 mm) 200 ms prior to the presynaptic Pyr doublet of spikes. Middle: example single traces showing the SST response to the first evoked Pyr spike under baseline/light OFF (black traces) and light ON (blue traces) conditions. Bottom: average responses from the above Pyr to SST connection (Light OFF, n=27 trials; Light ON, n=30 trials). (G) Mean EPSP amplitude in response to the first spike of the doublet, for both conditions. (H) Mean failure rate after the first spike, for both conditions. (I) and (J) The same as (G) and (H) but in the presence of the nicotinic receptors antagonist (mecamylamine). See also Figure S3 and S4.

Figure 7.. Presynaptic inhibition of PKA eliminates ACh-enhancement of Pyr-SST synapses.

(A) Schematic of the stimulation protocol. 1-single blue light (10 ms) was delivered 200 ms prior to the presynaptic spike train in the presence of PKA inhibitor peptide in the presynaptic Pyr cell. (B) The averaged trace of EPSP under baseline/light OFF and light ON conditions. Heatmaps at bottom show response amplitude for both conditions as described in previous figures. (C) Mean EPSP amplitude in response to the first spike in the train, for both conditions. (D) Mean failure rate after the first spike, for both conditions. For all, open circles represent individual cell measurements and filled circles represent all-cell mean± SEM, respectively (C,D). See also Figure S5.

Figure 8.. Pyr-PV and Pyr-Pyr connections are not enhanced during optogenetically driven ACh release.

(A) Schematic of the stimulation protocol for Pyr-PV connections. 1-single blue light (10 ms) was delivered 200 ms prior to the presynaptic spike train. (B) The averaged trace of EPSP under baseline/light OFF and light ON conditions. Heatmaps at bottom show response amplitude for both conditions as described in previous figures. (C) Mean EPSP amplitude in response to the first spike in the train, for both conditions. (D) Mean failure rate after the first spike, for both conditions. See also Figure S6. (E) Schematic of the stimulation protocol for L2 Pyr-Pyr connections. 1-single blue light (10 ms) was delivered 200 ms prior to the presynaptic spike train. (F) The averaged trace of 10 response trials for Pyr-Pyr connection under baseline condition (left) and in carbachol. Ten presynaptic spikes (dashed vertical lines) at 20 Hz were delivered. Heatmaps at bottom show response amplitude for 10, 10 spike trials, using a linear scale where red is maximum amplitude in both conditions. (G) Mean EPSP amplitude in response to the first spike in the train, for baseline/light OFF and light ON conditions. (H) Mean failure rate after the first spike, for both conditions. For all, open circles represent individual cell measurements and filled circles represent all-cell mean± SEM, respectively (C,D and G.H). See also Figure S7.