Extracellular hippocampal acetylcholine level controls amygdala function and promotes adaptive conditioned emotional response

Abstract

Ample data indicate that tone and contextual fear conditioning differentially require the amygdala and the hippocampus. However, mechanisms subserving the adaptive selection among environmental stimuli (discrete tone vs context) of those that best predict an aversive event are still elusive. Because the hippocampal cholinergic neurotransmission is thought to play a critical role in the coordination between different memory systems leading to the selection of appropriate behavioral strategies, we hypothesized that this cholinergic signal may control the competing acquisition of amygdala-mediated tone and contextual conditioning. Using pavlovian fear conditioning in mice, we first show a higher level of hippocampal acetylcholine release and a specific pattern of extracellular signal-regulated kinase 1/2 (ERK1/2) activation within the lateral (LA) and basolateral (BLA) amygdala under conditions in which the context is a better predictor than a discrete tone stimulus. Second, we demonstrate that levels of hippocampal cholinergic neurotransmission are causally related to the patterns of ERK1/2 activation in amygdala nuclei and actually determine the selection among the context or the simple tone the stimulus that best predicts the aversive event. Specifically, decreasing the hippocampal cholinergic signal not only impaired contextual conditioning but also mimicked conditioning to the discrete tone, both in terms of the behavioral outcome and the LA/BLA ERK1/2 activation pattern. Conversely, increasing this cholinergic signal not only disrupted tone conditioning but also promoted contextual fear conditioning. Hence, these findings highlight that hippocampal cholinergic neurotransmission controls amygdala function, thereby leading to the selection of relevant emotional information.

Figure 1.

Time course of ACh release in the hippocampus as a function of the conditioning procedure. Extracellular ACh variation is expressed in percentage of baseline levels (A) and in δ femtomoles per microliter (B; mean ± SEM) in animals submitted to either a CS–US pairing (n = 5) or CS–US unpairing procedure (n = 6).

Figure 2.

Effects of intrahippocampal infusions of SAL, SCOP, and PHY on cue tone and contextual fear conditioning as a function of the conditioning procedure. A, Auditory cue test. Conditioned response to the tone is expressed as percentage of freezing increase to the tone with respect to a baseline freezing level (i.e., pre-tone/post-tone periods mean). The ratio was calculated as follows: [% freezing during the tone presentation − (% pre-tone period freezing + % post-tone period freezing)/2]/[% freezing during the tone presentation + (% pre-tone period freezing + % post-tone period freezing)/2]. Values of this ratio (mean ± SEM) are illustrated for mice submitted to SAL (n = 21), SCOP (n = 19), or PHY (n = 19) intrahippocampal infusions before either a CS–US pairing or a CS–US unpairing procedure. B, Context test. Percentage of conditioned freezing (mean ± SEM) during the 4 min reexposure period in animals submitted to SAL, SCOP, or PHY intrahippocampal infusions before either a CS–US pairing or unpairing procedure. C, Scopolamine dose effect on conditioned freezing to the tone CS (mean ± SEM) in mice submitted to SAL (n = 7) or SCOP intrahippocampal infusions [20 (n = 8), 50 (n = 17), or 100 μg/μl (n = 8)] before a CS–US unpairing procedure. D, Scopolamine dose effect on conditioned freezing to the context (mean ± SEM) in animals submitted to SAL or SCOP intrahippocampal infusions (20, 50, or 100 μg/μl) before a CS–US unpairing procedure. E, Physostigmine dose effect on conditioned freezing to the tone CS (mean ± SEM) in mice submitted to SAL (n = 14) or PHY intrahippocampal infusions [0.02 (n = 9), 0.1 (n = 9), or 1 μg/μl (n = 8)] before a CS–US pairing procedure. F, Physostigmine dose effect on conditioned freezing to the context (mean ± SEM) in animals submitted to SAL or PHY intrahippocampal infusions (0.02, 0.1, or 1 μg/μl) before a CS–US pairing procedure. * indicates conditioning effect (CS–US pairing vs unpairing); # indicates pharmacological effect (SAL vs SCOP or SAL vs PHY).

Figure 3.

Specificity of the freezing response to the tone CS. A, Fear responses to the tone CS and to the neutral white noise (WN) is expressed as percentage of freezing increase to the tone, or to the white noise, with respect to a baseline freezing level (i.e., pre-tone/post-tone periods mean). Values of this ratio (mean ± SEM) are illustrated for SAL-infused mice submitted to a CS–US pairing procedure (n = 14) and for SCOP-infused mice submitted to an unpairing procedure (n = 17). B, Conditioned freezing responses to the white noise (WN) CS, expressed as percentage of freezing increase to the WN with respect to a baseline freezing level (mean ± SEM) in SAL-infused mice submitted to a WN–US pairing (n = 5) or unpairing procedure (n = 6). * indicates conditioning effect (CS–US pairing vs unpairing); # indicates test effect (tone CS vs white noise).

Figure 4.

Locomotor activity. Number of crossovers (mean ± SEM) during the first minute of the auditory cue test displayed by mice previously submitted to SAL, SCOP (50 μg/μl), or PHY (0.1 μg/μl) intrahippocampal infusions before either a CS–US pairing or unpairing procedure.

Figure 5.

Effects of intrahippocampal infusions of SAL (n = 10), SCOP (n = 10), and PHY (n = 10) on ERK1/2 phosphorylation in the hippocampus as a function of the conditioning procedure. A, Number of p-ERK1/2-immunopositive cells per square millimeter (mean ± SEM) in SAL, PHY, and SCOP mice submitted to either a CS–US pairing or unpairing procedure in DG and CA3 subregions of the dorsal (left) and ventral (right) hippocampus. B, Number of p-ERK1/2-immunopositive cells per square millimeter (mean ± SEM) in SAL, PHY, and SCOP mice submitted to either a CS–US pairing or unpairing procedure in CA1 in the dorsal (left) and ventral (right) part of the hippocampus. C, Representative photomicrographs depicting p-ERK1/2 levels within the CA1 subregion of the ventral hippocampus in SAL, PHY, and SCOP mice submitted to either a CS–US pairing (top) or unpairing (bottom) procedure. * indicates conditioning effect (CS–US pairing vs unpairing); # indicates pharmacological effect (SAL vs SCOP or SAL vs PHY)

Figure 6.

Effects of intrahippocampal infusions of SAL, SCOP, and PHY on ERK1/2 phosphorylation in the amygdala as a function of the conditioning procedure. A, Number of p-ERK1/2-immunopositive cells per square millimeter (mean ± SEM) in SAL, SCOP, and PHY mice trained with either a CS–US pairing or unpairing conditioning procedure in the LA and BLA nuclei of the amygdala. B, For each animal, the relative activity of each nucleus within the LA/BLA neural complex was calculated using the following ratio: (LA − BLA)/(LA + BLA). For each group, the mean ± SEM ratio is presented. C, Representative photomicrographs depicting p-ERK1/2 levels in the LA and BLA nuclei of the amygdala in SAL, PHY, and SCOP mice submitted to either a CS–US pairing (left) or unpairing (right) procedure. * indicates conditioning effect (CS–US pairing vs unpairing); # indicates pharmacological effect (SAL vs SCOP or SAL vs PHY)