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SummaryAcetylcholine (ACh) is released from basal forebrain cholinergic neurons in response to salient stimuli and engages brain states supporting attention and memory. These high ACh states are associated with theta oscillations, which synchronize neuronal ensembles. Theta oscillations in basolateral amygdala (BLA) underlie emotional memory, yet their mechanism remains unclear. Using brain slice electrophysiology in mice, we show large ACh stimuli evoke prolonged theta oscillations in BLA local field potential that depend upon activation of cholecystokinin (CCK) interneurons (INs). Somatostatin (SOM) INs inhibit CCK INs and are themselves inhibited by ACh, gating BLA theta. ACh-induced theta activity is more readily evoked in BLA over cortex or hippocampus, suggesting preferential activation of BLA during high ACh states. These data reveal a SOM-CCK IN circuit in BLA that gates internal theta oscillations and suggest a mechanism by which salient stimuli acting through ACh switch the BLA into a network state enabling emotional memory.

The amygdalar anterior basolateral nucleus (BLa) plays a vital role in emotional behaviors. This region receives dense cholinergic projections from basal forebrain which are critical in regulating neuronal activity in BLa. Cholinergic signaling in BLa has also been shown to modulate afferent glutamatergic inputs to this region. However, these studies, which have used cholinergic agonists or prolonged optogenetic stimulation of cholinergic fibers, may not reflect the effect of physiological acetylcholine release in the BLa. To better understand these effects of acetylcholine, we have used electrophysiology and optogenetics in male and female mouse brain slices to examine cholinergic regulation of afferent BLa input from cortex and midline thalamic nuclei. Phasic ACh release evoked by single pulse stimulation of cholinergic terminals had a biphasic effect on transmission at cortical input, producing rapid nicotinic receptor-mediated facilitation followed by slower mAChR-mediated depression. In contrast, at this same input, sustained ACh elevation through application of the cholinesterase inhibitor physostigmine suppressed glutamatergic transmission through mAChRs only. This suppression was not observed at midline thalamic nuclei inputs to BLa. In agreement with this pathway specificity, the mAChR agonist, muscarine more potently suppressed transmission at inputs from prelimbic cortex than thalamus. Muscarinic inhibition at prelimbic cortex input required presynaptic M4 mAChRs, while at thalamic input it depended on M3 mAChR-mediated stimulation of retrograde endocannabinoid signaling. Muscarinic inhibition at both pathways was frequency-dependent, allowing only high-frequency activity to pass. These findings demonstrate complex cholinergic regulation of afferent input to BLa that is pathway-specific and frequency-dependent.SIGNIFICANCE STATEMENTCholinergic modulation of the basolateral amygdala regulates formation of emotional memories, but the underlying mechanisms are not well understood. Here, we show, using mouse brain slices, that ACh differentially regulates afferent transmission to the BLa from cortex and midline thalamic nuclei. Fast, phasic ACh release from a single optical stimulation biphasically regulates glutamatergic transmission at cortical inputs through nicotinic and muscarinic receptors, suggesting that cholinergic neuromodulation can serve precise, computational roles in the BLa. In contrast, sustained ACh elevation regulates cortical input through muscarinic receptors only. This muscarinic regulation is pathway-specific with cortical input inhibited more strongly than midline thalamic nuclei input. Specific targeting of these cholinergic receptors may thus provide a therapeutic strategy to bias amygdalar processing and regulate emotional memory.

The infrequency of a total solar eclipse renders the event novel to those animals that experience its effects and, consequently, may induce anomalous behavioral responses. However, historical information on the responses of animals to eclipses is scant and often conflicting. In this study, we qualitatively document the responses of 17 vertebrate taxa (including mammals, birds, and reptiles) to the 2017 total solar eclipse as it passed over Riverbanks Zoo and Garden in Columbia, South Carolina. In the days leading up to the eclipse, several focal teams, each consisting of researchers, animal keepers, and student/zoo volunteers conducted baseline observations using a combination of continuous ad libitum and scan sampling of each animal during closely matched seasonal conditions. These same focal teams used the same protocol to observe the animals in the hours preceding, during, and immediately following the eclipse. Additionally, for one species&mdash, siamangs (Symphalangus syndactylus)&mdash, live video/audio capture was also employed throughout observations to capture behavior during vocalizations for subsequent quantitative analysis. Behavioral responses were classified into one or more of four overarching behavioral categories: normal (baseline), evening, apparent anxiety, and novel. Thirteen of seventeen observed taxa exhibited behaviors during the eclipse that differed from all other observation times, with the majority (8) of these animals engaging in behaviors associated with their evening or nighttime routines. The second predominant behavior was apparent anxiety, documented in five genera: baboons (Papio hamadryas), gorillas (Gorilla gorilla gorilla), giraffes (Giraffa cf. camelopardalis), flamingos (Phoenicopterus ruber), and lorikeets (Trichoglossus moluccanus and Trichoglossus haematodus). Novel behaviors characterized by an increase in otherwise nearly sedentary activity were observed only in the reptiles, the Galapagos tortoise (Chelonoidis nigra) and the Komodo dragon (Varanus komodoensis). While the anthropogenic influences on animal behaviors&mdash, particularly those relating to anxiety&mdash, cannot be discounted, these observations provide novel insight into the observed responses of a diverse vertebrate sample during a unique meteorological stimulus, insights that supplement the rare observations of behavior during this phenomenon for contextualizing future studies.

Although it is known that acetylcholine acting through M1 muscarinic receptors (M1Rs) is essential for memory consolidation in the anterior basolateral nucleus of the amygdala (BLa), virtually nothing is known about the circuits involved. In the hippocampus M1R activation facilitates long-term potentiation (LTP) by potentiating NMDA glutamate receptor (NMDAR) currents. The majority of NMDAR+ profiles in the BLa are spines. Since about half of dendritic spines of BLa pyramidal neurons (PNs) receiving glutamatergic inputs are M1R-immunoreactive (M1R+) it is possible that the role of M1Rs in BLa mnemonic functions also involves potentiation of NMDAR currents in spines. However, the finding that only about half of BLa spines are M1R+ suggests that this proposed mechanism may only apply to a subset of glutamatergic inputs. As a first step in the identification of differential glutamatergic inputs to M1R+ spines in the BLa, the present electron microscopic study used antibodies to two different vesicular glutamate transporter proteins (VGluTs) to label two different subsets of glutamatergic inputs to M1R+ spines. These inputs are largely complimentary with VGluT1+ inputs arising mainly from cortical structures and the basolateral nucleus, and VGluT2+ inputs arising mainly from the thalamus. It was found that about one-half of the spines that were postsynaptic to VGluT1+ or VGluT2+ terminals were M1R+. In addition, a subset of the VGluT1+ or VGluT2+ axon terminals were M1R+, including those that synapsed with M1R+ spines. These results suggest that acetylcholine can modulate glutamatergic inputs to BLa spines by presynaptic as well as postsynaptic M1R-mediated mechanisms.