No abstract

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 (MTN). 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 muscarinic receptor (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 MTN inputs to BLa. In agreement with this pathway-specificity, the mAChR agonist, muscarine more potently suppressed transmission at inputs from prelimbic cortex (PL) than thalamus. Muscarinic inhibition at PL input required presynaptic M4 mAChRs, while at thalamic input it depended upon 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 statement:Cholinergic 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 MTN. 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 MTN input. Specific targeting of these cholinergic receptors may thus provide a therapeutic strategy to bias amygdalar processing and regulate emotional memory.

As the inhibitory γ-aminobutyric acid–ergic (GABAergic) transmission has a pivotal role in the central nervous system (CNS) and defective forms of its synapses are associated with serious neurological disorders, numerous versions of caged GABA and, more recently, photoswitchable ligands have been developed to investigate such transmission. While the complementary nature of these probes is evident, the mechanisms by which the GABA receptors can be photocontrolled have not been fully exploited. In fact, the ultimate need for specificity is critical for the proper synaptic exploration. No caged allosteric modulators of the GABAA receptor have been reported so far; to introduce such an investigational approach, we exploited the structural motifs of the benzodiazepinic scaffold to develop a photocaged version of diazepam (CD) that was tested on basolateral amygdala (BLa) pyramidal cells in mouse brain slices. CD is devoid of any intrinsic activity toward the GABAA receptor before irradiation. Importantly, CD is a photoreleasable GABAA receptor-positive allosteric modulator that offers a different probing mechanism compared to caged GABA and photoswitchable ligands. CD potentiates the inhibitory signaling by prolonging the decay time of postsynaptic GABAergic currents upon photoactivation. Additionally, no effect on presynaptic GABA release was recorded. We developed a photochemical technology to individually study the GABAA receptor, which specifically expands the toolbox available to study GABAergic synapses.

Acetylcholine (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 and synaptic transmission. Cholinergic signaling in BLa is thought to occur through both a slow mode of volume transmission as well as a rapid, phasic mode. However, the relative effect of each mode of signaling in BLa is not understood. Here, we used electrophysiology and optogenetics in mouse brain slices to compare regulation of afferent input from cortex and thalamus to the BLa by these two modes of transmission. Phasic ACh release evoked by single pulse stimulation of cholinergic terminals had a biphasic effect on glutamatergic transmission at cortical input, producing rapid nicotinic receptor-mediated facilitation followed by slower muscarinic receptor (mAChR)-mediated depression. In contrast, tonic elevation of ACh through application of the cholinesterase inhibitor physostigmine suppressed glutamatergic transmission at cortical inputs through mAChRs only. This suppression was not observed at thalamic inputs to BLa. In agreement with this pathway-specificity, the mAChR agonist, muscarine more potently suppressed transmission at inputs from prelimbic cortex (PL) than thalamus. Muscarinic inhibition at PL input was dependent on presynaptic M4 mAChRs, while at thalamic input it depended upon 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 depends upon the mode of ACh release and is both pathway specific and frequency dependent.