Olfactory bulb acetylcholine release dishabituates odor responses and reinstates odor investigation

Abstract

Habituation and dishabituation modulate the neural resources and behavioral significance allocated to incoming stimuli across the sensory systems. We characterize these processes in the mouse olfactory bulb (OB) and uncover a role for OB acetylcholine (ACh) in physiological and behavioral olfactory dishabituation. We use calcium imaging in both awake and anesthetized mice to determine the time course and magnitude of OB glomerular habituation during a prolonged odor presentation. In addition, we develop a novel behavioral investigation paradigm to determine how prolonged odor input affects odor salience. We find that manipulating OB ACh release during prolonged odor presentations using electrical or optogenetic stimulation rapidly modulates habituated glomerular odor responses and odor salience, causing mice to suddenly investigate a previously ignored odor. To demonstrate the ethological validity of this effect, we show that changing the visual context can lead to dishabituation of odor investigation behavior, which is blocked by cholinergic antagonists in the OB.

Fig. 1

Glomerular responses to a prolonged odor presentation habituate over time in anesthetized mice. a Pseudo-color glomerular responses to 2-heptanone (0.3% s.v.) initially and after 30 s of the odor presentation. b The average fluorescence trace (% of initial response ± s.e.m.) from all recorded glomeruli (n = 96, 6 mice). c The average glomerular odor response (% of initial response ± s.e.m) decreases during the odor presentation. *p < 0.05

Fig. 2

Electrical basal forebrain stimulation (BFS) dishabituates glomerular odor responses in anesthetized mice. a Schematic illustration of the imaging experiment. b Pseudo-color glomerular responses to 2-heptanone (0.5% s.v.) initially and before and after BFS (50 µA, 3 s) at 50 and 5 Hz. c Fluorescence traces (% of initial response) from the glomerulus indicated by arrows in b. d The average glomerular odor response (% of initial response ± s.e.m) decreases during the odor presentation and increases following 50 Hz BFS (n = 76, 5 mice). e In a subset of mice, 5 Hz stimulation was given in addition to 50 Hz, and it did not increase the average glomerular odor response. *p < 0.05. Illustration by M.C.O.

Fig. 3

Olfactory investigation behavior increases with odor onset and habituates over time. a Investigation time (seconds) is not affected when odor is not present, but b increases with odor (10% ethyl butyrate) onset (n = 10 mice). c Investigation time in control mice increases with odor onset and decreases over the exposure (n = 10 mice). Error bars = s.e.m., *p < 0.05

Fig. 4

Optogenetic light stimulation (OLS) can dishabituate odor investigation behavior. a Schematic illustration of the OLS experiment. b Investigation time (seconds ± s.e.m.) does not increase after 50 Hz OLS in WT (ChR2−) controls (n = 5 mice). c Raster plots of the odor investigation behavior comparing OLS at 5, 25, and 50 Hz in the same mice (n = 5 mice). d Investigation time in Ch2R+ mice after the OLS is stimulation dependent: following 50 Hz OLS, investigation time increases and stays increased for at least 3 min. After 25 HZ OLS, investigation time also increases, but does not remain increased. 5 Hz OLS does not increase investigation time. e Raster plots of investigation behavior taken from two mice before and after OLS (blue line) in the absence of any odor stimuli demonstrating the OLS itself does not lead to increased sniffing. f Mean investigation times (seconds ± s.e.m.) taken from the minute before and after OLS in mice in the absence odor stimulation. *p < 0.05. Illustration by Ogg, M.C.

Fig. 5

A visual context change (VS) dishabituates odor investigation behavior. a Schematic illustration of the VS experiment. b Investigation time (seconds ± s.e.m.) after the VS does not increase if odor is not present (n = 5 mice). c Raster plots of the odor investigation behavior following olfactory bulb (OB) cannula infusion of vehicle (Ringer’s) or the cholinergic antagonist scopolamine (n = 10 mice). d Investigation time after the VS increases when mice receive an OB cannula infusion of Ringer’s solution, but does not increase when the mice receive scopolamine. Illustration by Ogg, M.C.

Fig. 6

A visual context change (VS) dishabituates glomerular odor responses following short and long odor presentations in awake mice. a Average fluorescence trace (% of initial response ± s.e.m.) from all recorded glomeruli during control (top trace; n = 96 glomeruli, 4 mice) and VS conditions (white box, bottom trace; n = 73 glomeruli, 4 mice). b During 30 s trials, the average glomerular odor response (% of initial response ± s.e.m.) decreases during the odor presentation and significantly increases following VS. c During 7 min trials, the average glomerular odor response (% of initial response ± s.e.m.) decreases during the odor presentation (n = 92 glomeruli, 4 mice) and significantly increases following VS (n = 86 glomeruli, 4 mice)