Differential Regulation of Prelimbic and Thalamic Transmission to the Basolateral Amygdala by Acetylcholine Receptors

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.

Acetylcholine engages distinct amygdala microcircuits to gate internal theta rhythm

“…Accordingly, external glutamatergic inputs, such as those from cortex, could drive synchronization in BLA through interaction with PV INs. Alternatively, ACh suppresses cortical glutamatergic input 52 and promotes a network state in BLA in which CCK INs internally drive theta oscillations without the need for external glutamatergic input. This suggests externally and internally driven theta oscillations in the BLA could be generated by these competing IN networks.…”

“…Indeed, PV INs have been implicated in BLA theta oscillations in some 48–50 , but not all conditions 51 . Alternately, ACh generates theta oscillation in the BLA in vivo , but also inhibits glutamate release from afferent cortical input 52 . This suggests favorable conditions for internally generated BLA oscillations.…”

“…PV INs in BLA are strongly driven by glutamatergic input, providing a possible mechanism for external glutamatergic control of theta oscillations 47 . Indeed, PV INs have been implicated in BLA theta oscillations in some [48][49][50] , but not all conditions 51. Alternately, ACh generates theta oscillation in the BLA in vivo, but also inhibits glutamate release from afferent cortical input 52 . This suggests favorable conditions for internally generated BLA oscillations.…”

ChAT::Cre transgenic rats show sex‐dependent altered fear behaviors, ultrasonic vocalizations and cholinergic marker expression

“…Immunofluorescence labeling for ChAT and VAChT used methods described in Tryon et al, (2022). 47 For ChAT immunolabeling, sections were washed three times in Tris buffer (TBS) for 10 min, blocked in TBS containing 0.5% Triton X-100 and 10% normal donkey serum for 30 min at room temperature, washed in TBS then incubated with goat polyclonal anti-choline acetyltransferase antibodies (1:1000; Millipore Cat# AB144P, RRID:AB_2079751;used previously in 47 ) in TBS containing 0.5% Triton X-100 and 2% normal donkey serum for 2 days at room temperature. Sections were then incubated in donkey-anti goat conjugated to Alexa Fluor 647 (1:400; Jackson ImmunoResearch Labs Cat# 705-605-147, RRID:AB_2340437) in TBS with 0.5% Triton X- Coronal sections including the BF, BLA and hippocampus were also stained for acetylcholinesterase (ACHE) activity.…”

“…Immunofluorescence labeling for ChAT and VAChT used methods described in Tryon et al, (2022). 47 For ChAT immunolabeling, sections were washed three times in Tris buffer (TBS) for 10 min, blocked in TBS containing 0.5% Triton X-100 and 10% normal donkey serum for 30 min at room temperature, washed in TBS then incubated with goat polyclonal anti-choline acetyltransferase antibodies (1:1000; Millipore Cat# AB144P, RRID:AB_2079751;used previously in 47 ) in TBS containing 0.5% Triton X-100 and 2% normal donkey serum for 2 days at room temperature.…”