Acetylcholine in learning and memory

National Institute Of Neurological Disorders And Stroke

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Abstract

All projects developed under this Z01 involved extensive collaborations with Dr. D. Talmage (NINDS) and several members of his laboratory (Kim, Jiang & Rajebhosale). Some of this work also employed the expertise of Dr Corey Johnson (NINDS Bioinformatics core). Overall, our functional mapping of cholinergic neurons engaged in memory-encoding has focused on in-depth analyses of the basal forebrain cholinergic projection neurons (BFCNs) associated with (1) aversive vs. appetitive memories and (2) circuits involved in innate vs cue-associated learning. BFCNs are organized along the rostro-caudal axis of the adult mammalian forebrain, and include the medial septum (MS), diagonal band (vertical and horizontal limbs; vDB hDB), the substantia innominata (SI), ventral pallidum (VP/Sia), and nucleus basalis of Meynert (NBM/Sip). These cholinergic projection neurons participate in an array of cognitive functions through the action of acetylcholine released at pre- and post-synaptic sites within multiple cortical and subcortical regions. Recent studies indicate that key determinants of BFCNs include birth order and location; specifically, early-born cholinergic neurons are more caudally located, in contrast to later-born rostral BFCNs (e.g., Ananth et al., Nat. Rev. Neurosci., 2023). However, the distinctions amongst BFCNs in inputs and targets, physiology and morphology, genetic profiles and resilience (or lack thereof) to degeneration remain unknown. A. Transcriptomic diversity of BFCNs: Current studies expand the analysis of BFCNs from a focused dissection of inputs and targets, as well as physiological and morphological features, to include genetic profiles by transcriptomic analysis of all basal forebrain cholinergic nuclei. We have gathered data from over 20,000 identified cholinergic nuclei (using a nuclear marker genetic line and crossing this with a cholinergic CRE line) from young animals. These studies, which are in preparation for publication (Ananth et al 2025 in prep), reveal a startling diversity of cholinergic neurons with more than 20 separately identified transcriptomic clusters. Assessment of the overlap of transcriptomic clusters vs classic anatomical designations reveals that each of the anatomically defined regions (i.e. MS, DBB, SI, VP and NBM ; as defined above) are spread across at multiple distinct clusters. Studies are underway to assess the relationship between these transcriptomic subtypes and the complex arrays of physiological properties that we have now established using a similar unsupervised sorting (UMAP) assessment of 18 electrophysiological characteristics (Watkins & Luo et al, 2026 in prep). B. Physiological diversity of BFCNs and the effects of learned experience on electrophysiological profile. BFCNs differ in multiple characteristics pertaining to their overall excitability that are readily separable from one another by unsupervised clustering algorithms (Watkins, Luo et al 2026a, in prep) Perhaps even more intriguing are our recent findings demonstrating differential effects of learned experience on distinct aspects of excitability, consistent with the idea that specific subsets of neurons appear to encode different aspects of learning. For example, using a combination of activity-dependent genetic markers and ex vivo electrophysiological analyses we have found multiple populations of BFCNs that are engaged in threat learning but that are differentially affected by threat training vs recall. Furthermore, the initial changes in channel properties that underlie the increased excitability profile detected with initial recall relax back to pre-experience profiles over the next 3 days, when differences in the basic physiological properties are "replaced" by differences in synaptic input. Thus the encoding of experience transitions from short term changes in basic properties to longer-term changes in synaptic E/I balance, consistent with an important role of synaptic homeostasis. C: NBM/SIp cholinergic neurons are required for the encoding of cue-associated threat learning. (Rajebhosale & Ananth et al, 2024). Using a combination of genetic, immunological and retrograde markers to identify specific BFCNs, their projection targets, and their engagement in memory, we were able to functionally map the activation (or lack thereof) of BFCNs during different phases of learning and /or whether the BFCNs are reactivated by memory retrieval. We now have several lines of evidence demonstrating that distinct subsets of basal forebrain cholinergic projection neurons are requisite partners in the establishment of engrams that encode innate vs. learned threat memory. With genetically encoded ACh sensors in the basal lateral amygdala (BLA), we demonstrate that BLA-projecting cholinergic neurons can learn the association between a naive tone and a shock, as manifest in enhanced ACh release in response to the conditioned tone alone, after 24 hours ( and for multiple days) after the initial training. Cholinergic neurons of the NBM/SIp manifest immediate early gene responses and increased intrinsic excitability following the tone-elicited memory response. Silencing these cue-associated, engram-enrolled, cholinergic neurons prevents expression of the defensive behavior to the tone. In contrast, silencing ventral pallidal and anterior SI (VP/SIa) cholinergic neurons, the other major source of cholinergic input to the BLA, does not alter cue-associated learning. Instead, VP/SIa cholinergic neurons are activated in response to innate threat (predator odor), a stimulus that does not activate NBM/SIp cholinergic neurons. Taken together, these studies reveal that distinct populations of cholinergic neurons are recruited to signal distinct aversive stimuli, demonstrating functionally refined organization of specific types of memory within the cholinergic basal forebrain. D. Unique subpopulations of VP/SIa cholinergic projection neurons encode innate responses to opposite valence olfactory cues. (Kim et al.,2024; Kim et al 2025 in prep.) We next pursued the mechanism of cholinergic engagement in innate learning with both aversive and appetitive stimuli in a closer examination of the VP/SIa cholinergic projection neurons. Using intersectional genetics combined with behavioral performance tests, plus in vivo assays of Ca signaling and ACh release, we examined the activation profile of VP cholinergic neurons in response to an appetitive vs an aversive odor. First, VP cholinergic neurons were engaged in innate behavioral responses to each odor: appetitive odors elicited approach behavior while aversive odors led to avoidance behavior. Activity and cre-dependent viral vectors revealed that these behaviors engage two distinct, non-overlapping subpopulations of VP cholinergic neurons, depending on the valence of the odor stimulus (App vs Avers): The two subpopulations of cholinergic neurons are physically intermingled within the VP, but show differences in specific aspects of their electrophysiological and morphological profiles. Finally, Appetitive-encoding VP cholinergics differ from aversive-encoding VP cholinergics in the relative representation of their projections to the basolateral amygdala, and in the behavioral responses to selective inhibition. Our results highlight the functional heterogeneity of cholinergic neurons within the VP in demonstrating their distinct valence encoding profile and their differential role in innate motivated behaviors. Ongoing work in VP combines in vivo endoscopic Ca, ACh sensor and optogenetic stimulation of aversive vs appetitive populations in their projections to BLA. Comparing VP and NBM cholinergics in our genetic/activity labeling plus electrophysiological studies shows that, despite the fact that both the VP and NBM strongly innervate the BLA, they are largely distinct in their intrinsic properties, reflecting their very distinct engagement in innate vs cue-associated behaviors. Furthermore, short-term plastic changes in the electrophysiological profile that are transiently induced by RECALL in NBM, cue-associated cholinergic engram neurons, target the same properties that distinguish them, at rest, from VP cholinergic neurons. Hence the encoding of state in distinct learning paradigms may be key to plastic changes in the electrophysiological profile of cholinergic engram neurons. E. Other ongoing studies on ACh signaling and memory encoding examine (1) the role of cholinergic inputs in behaviors involving specific hippocampal and cortical targets (Entorhinal cortex , Prefrontal cortex, DG /CA2/3 ) (2) expands the AP axis of BFCNs studied (MS, hDB), (3) examines the profile of cholinergic axonal excitability & the differential release of ACh along extensive axonal projections and (4) examines the developmental profile and NRG dependence of cholinergic projections engaged in memory encoding, retention and extinction . F. Last ( but not least) Methods developed in the course of this research - in particular on Ca imaging algorithms -- have also been reported in publication (Desai et al., 2024) These have all been entered into data sharing sites, as required.