Acetylcholine in learning and memory

National Institute Of Neurological Disorders And Stroke

Investigators

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Abstract

2022/3 was the first year our colony reached its full repertoire of genetically modified mice. With these in hand and our new viral tools developed for intersectional genetic approaches, the work assessing the role of cholinergic signaling in memory took off full blast. All projects developed under this Z01 involved extensive collaborations with Dr. D. Talmage (NINDS) . Some of this work was also the beneficiary of the expertise of Dr. Fields (NINDS Viral Core) and Dr 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, i.e., early-born cholinergic neurons are more caudally located, in contrast to later born rostral BFCNs (see Ananth et al. Nat. Rev. Neurosci, 2023). However, the distinctions amongst BFCNs in inputs and targets, physiology and morphology, genetic profiles and vulnerability (or lack thereof) to degeneration remain unknown. A: NBM/SIp cholinergic neurons participate in the encoding of cue-associated threat learning. (Rajebhosale & Ananth et al, 2023 eLife) 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 engrams that encode innate vs. learned threat memory. Using a genetically encoded ACh sensor in the basal lateral amygdala (BLA), we show that BLA-projecting cholinergic neurons can learn the association between a nave tone and a shock as manifest in enhanced ACh release in response to conditioned tone alone, 24h after 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 second 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. B. Unique subpopulations of VP/SIa cholinergic projection neurons encode innate responses to opposite valence olfactory cues. (Kim et al., 2023 sub. to Neuron; Kim et al 2023 in prep. for JNS) 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: the appetitive odor elicited approach-behavior while an aversive odor 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, App. VP cholinergics differ from Avers. 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. 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 engagement in innate vs cue-associated behaviors. Furthermore, short-term plastic changes in electrophysiological profile that are transiently induced by RECALL in NBM, cue-associated cholinergic engram neurons, target the same set of properties that distinguish them from VP cholinergic neurons that encode innate learning. Hence the encoding of state in distinct learning paradigms may be key to plastic changes in the electrophysiological profile of cholinergic engram neurons. C. Comparative analysis of BLA- projecting cholinergic neurons in NBM/SI of mouse vs macaque (Luo et al, in prep) Thanks to the NHPCC (a consortium of investigators from 5 different neuro ICs that work together to maximize utilization of macaque tissue; see IRP collaborators) we have been able to extend our analysis to a rigorous comparison of the morphoelectric profiles of mouse vs. macaque BLA-projecting, NBM/ SI, cholinergic neurons. This work uses a combination of retrograde labeling from BLA with beads, and BFCN identification with a cholinergic enhancer virus (Fishell, Harvard U.) for live labeling and recording. These studies were followed up with biocytin +ChAT IHC for post hoc re-localization and proximal arbor reconstruction of over 100 mouse vs macaque, BLA projecting, cholinergic NBM neurons. Surprisingly, these acute slice recording studies of these functionally analogous populations revealed striking species differences. Future studies in marmosets where we will be able to better control the experience profile of the animals prior to sampling may shed light on whether the distinguishing features are also a function of state. D. Other ongoing studies on ACh signaling and memory encoding examine (1) the role of specific targets ( i.e. vHipp vs BLA; in prep: Wang et al., Zhong et al., Watkins et al.) (2) expands the AP axis of BFCNs studied (MS, DB; in prep Zhong et al., Watkins et al.), (3) examines the profile of cholinergic axonal excitability & effects of presynaptic modulators (Zhong et al., Desai et al., and (4) examine the developmental profile of cholinergic projections engaged in memory encoding, retention and extinction (Jiang et al., in prep). A new collaboration with Dr Julius Zhu, at UVA will further our studies on high resolution imaging of nanoscale assays of ACh release.