Olfactory Bulb Muscarinic Acetylcholine Type 1 Receptors Are Required for Acquisition of Olfactory Fear Learning

Abstract

The olfactory bulb (OB) receives significant cholinergic innervation and widely expresses cholinergic receptors. While acetylcholine (ACh) is essential for olfactory learning, the exact mechanisms by which ACh modulates olfactory learning and whether it is specifically required in the OB remains unknown. Using behavioral pharmacology and optogenetics, we investigated the role of OB ACh in a simple olfactory fear learning paradigm. We find that antagonizing muscarinic ACh receptors (mAChRs) in the OB during fear conditioning but not testing significantly reduces freezing to the conditioned odor, without altering olfactory abilities. Additionally, we demonstrate that m1 mAChRs, rather than m2, are required for acquisition of olfactory fear. Finally, using mice expressing channelrhodopsin in cholinergic neurons, we show that stimulating ACh release specifically in the OB during odor-shock pairing can strengthen olfactory fear learning. Together these results define a role for ACh in olfactory associative learning and OB glomerular plasticity.

Keywords: acetylcholine; behavior; fear learning; muscarinic; olfaction; olfactory bulb; pharmacology.

Figure 1

Direct olfactory bulb (OB) application of scopolamine (SCOP) during fear conditioning impairs olfactory aversive fear learning but has no effect on the expression of previously learned fear. (A) Mice received infusions of vehicle (VEH) or different concentrations of SCOP (1 μM, 1 mM, or 10 mM), a non-selective antagonist of muscarinic acetylcholine receptors (mAChRs), through cannula directly into the OBs prior to olfactory fear conditioning, in which a single odor (E5) was paired with mild foot shock. Mice were tested for behavioral freezing to the conditioned odor (E5) 24 h later. Mice receiving infusions of 1 mM and 10 mM SCOP demonstrated reduced freezing relative to VEH controls, indicating impaired fear learning when mAChRs are blocked specifically in the OBs. (B) Mice were first fear-conditioned to E5. During testing, 24 h after conditioning, mice received direct OB infusions of VEH or 1 mM SCOP. There is no significant difference in behavioral freezing between mice receiving infusions of VEH or 1 mM SCOP, signifying antagonism of mAChRs during expression does not affect olfactory perception or behavioral displays of learned olfactory fear. Data presented as mean ± SEM. *p < 0.05.

Figure 2

Inhibition of mAChR1, but not mAChR2, decreases behavioral freezing to the conditioned odor. Mice received direct OB infusions of vehicle [VEH, either Ringer’s or dimethyl sulfoxide (DMSO)] or AFDX, a specific antagonist of the m2 subtype of mAChRs, or PIR, a specific antagonist of the m1 subtype of mAChRs, prior to olfactory fear conditioning. The mice were then tested for behavioral freezing 24 h later. (A) Mice receiving Ringer’s VEH and those receiving DMSO VEH prior to conditioning do not exhibit different freezing during testing, indicating no difference in learning as a result of the different VEH conditions. (B) There is no significant difference in freezing between VEH mice (combined Ringer’s and DMSO) and those receiving infusions of the mAChR2 antagonist AFDX; however, mice treated with PIR before conditioning display reduced freezing relative to VEH mice, suggesting mAChR1 s specifically are required for appropriate acquisition of olfactory fear. Data presented as mean ± SEM. *p < 0.05.

Figure 3

Enhanced OB ACh during odor-shock pairing augments olfactory fear learning. Mice were surgically implanted with a miniature LED directly above the OBs. During olfactory fear conditioning, all mice received light stimulation at the end of each of the six odor-shock pairings. Positive ChAT-ChR2 mice express channelrhodopsin in cholinergic cell populations, such that light stimulation should induce release of ACh in the OBs during odor-shock pairing. When tested 24 h later, ChAT-ChR2+ mice freeze significantly more than ChAT-ChR2− mice, which do not express channelrhodopsin in cholinergic cell populations and should experience no additional ACh release in the OBs as a result of light stimulation. This suggests that increasing OB ACh during olfactory fear conditioning can strengthen fear learning. Data presented as mean ± SEM. *p < 0.05.

Figure 4

Antagonism of mAChRs does not alter olfactory-driven behaviors. Mice underwent an olfactory investigation paradigm inside an open field chamber to determine whether the mAChR antagonist, SCOP, alters olfactory behaviors. Cannulated mice received direct OB infusions of either 1 mM SCOP or VEH. Time spent performing olfactory investigative behaviors was then scored in response to uncued odor presentations. Mice receiving OB SCOP did not differ from those receiving VEH in terms of time spent investigating. Together, this demonstrates non-specific antagonism of mAChRs does not alter olfactory-driven behaviors or induce temporary anosmia. Data presented as mean ± SEM.