Peripheral CB1 receptor blockade acts as a memory enhancer through a noradrenergic mechanism

Abstract

Peripheral inputs continuously shape brain function and can influence memory acquisition, but the underlying mechanisms have not been fully understood. Cannabinoid type-1 receptor (CB1R) is a well-recognized player in memory performance, and its systemic modulation significantly influences memory function. By assessing low arousal/non-emotional recognition memory in mice, we found a relevant role of peripheral CB1R in memory persistence. Indeed, the peripherally-restricted CB1R specific antagonist AM6545 showed significant mnemonic effects that were occluded in adrenalectomized mice, and after peripheral adrenergic blockade. AM6545 also transiently impaired contextual fear memory extinction. Vagus nerve chemogenetic inhibition reduced AM6545-induced mnemonic effect. Genetic CB1R deletion in dopamine β-hydroxylase-expressing cells enhanced recognition memory persistence. These observations support a role of peripheral CB1R modulating adrenergic tone relevant for cognition. Furthermore, AM6545 acutely improved brain connectivity and enhanced extracellular hippocampal norepinephrine. In agreement, intra-hippocampal β-adrenergic blockade prevented AM6545 mnemonic effects. Altogether, we disclose a novel CB1R-dependent peripheral mechanism with implications relevant for lengthening the duration of non-emotional memory.

Fig. 1. Pharmacological or genetic inhibition of CB1R improves memory persistence in the novel object-recognition test (NORT).

A Discrimination index values obtained at 3 h, 24 h, and 48 h after the training phase (n = 5–8). Discrimination index values in NORT at 48 h (B) after acute post-training treatment with vehicle (VEH) or rimonabant (RIM) (1 mg/kg) (n = 7–11), C in CB1HZ and WT mice (n = 6–8), after acute post-training treatment with vehicle (VEH) or AM6545 (1 mg/kg) in D CD-1 (n = 13 - 14) and E C57BL/6 J (n = 6, 7). F Discrimination index values in NORT at 24 h after acute post-training treatment with vehicle (VEH) or AM6545 (1 mg/kg) with a 3 min training period (n = 9–10). G Percentage of freezing in the context fear conditioning across extinction sessions (Ext1-Ext4) after acute post-extinction 1 treatment with vehicle (VEH) or AM6545 (1 mg/kg) (n = 12). Data are expressed as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA or two-way repeated measures ANOVA test followed by Bonferroni post hoc or Student’s t-test.

Fig. 2. AM6545 enhances novel object-recognition memory through a peripheral β-adrenergic mechanism.

A Discrimination index values obtained in the NORT performed at 48 h of adrenalectomized (ADX) or naive mice treated with vehicle (VEH) or AM6545 (1 mg/kg) (n = 6–8). B Schematic representation of pre-treatment and treatment after the training phase. Discrimination index values obtained in the NORT performed at 48 h of mice treated with vehicle (VEH) or AM6545 (1 mg/kg) after pre-treatment with C vehicle (VEH) or mifepristone (50 mg/kg) (n = 6–7) and D saline or sotalol (10 mg/kg) (n = 8–10). E Discrimination index values in WT or DBH-CB1KO mice in NORT at 48 h F and after saline or sotalol (10 mg/kg) treatment (n = 6–8). G Discrimination index values for mice pre-treated with saline or sotalol (10 mg/kg) prior to rimonabant (RIM) (1 mg/kg) or vehicle (VEH) in the NORT at 48 h (n = 9–11). Data are expressed as mean ± s.e.m. *p < 0.05 (treatment effect) ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 (pre-treatment effect) by two-way ANOVA test followed by Tukey post hoc or Student’s t-test.

Fig. 3. AM6545 memory improvement depends on vagus nerve activation.

A Glucose blood levels for 120 min after acute vehicle (VEH) or AM6545 (1 mg/kg) administration in mice (n = 6–7). B Discrimination index values in NORT at 48 h after acute vehicle (VEH) or AM6545 (1 mg/kg) and CNO (3 mg/kg) administration in mice infected with AAV5-mCherry-control in the vagus nerve (n = 6–7). C Discrimination index values in NORT at 48 h after acute vehicle (VEH) or AM6545 (1 mg/kg) and VEH or CNO (3 mg/kg) administration in mice infected with AAV5-mCherry-hM4Di in the vagus nerve (n = 8–12). Data are expressed as mean ± s.e.m. For the blood glucose measures statistical significance was calculated by two-way repeated measures ANOVA test. For NORT *p < 0.05 by Student’s t-test or two-way ANOVA test.

Fig. 4. Central effects of AM6545 administration on c-Fos expression and brain functional connectivity by resting state functional magnetic resonance imaging (rsfMRI).

Network graphs of c-Fos expression based on the Pearson coefficient in vehicle (A) and AM6545 (1 mg/kg) (B) between different brain areas, including prelimbic cortex (PL), infralimbic cortex (IL), cornu ammonis 1 (CA1), dentate gyrus (DG), cornu ammonis 3 (CA3), locus coeruleus (LC), basal amygdala (BA), central amygdala (CeA) and lateral amygdala (LA) (n = 5–9). Significant correlations are depicted with black lines. Z-score values of the positive (C) and negative (D) Pearson r correlation coefficients between the different brain regions analysed. E Whole brain local efficiency and nodal efficiencies of left frontal (F) cortex, right hippocampus (G) and right globus pallidus (H) (n = 10). I Statistical map showing significant differences between connectivity of posterior brainstem with the rest of the brain in vehicle and AM6545-treated animals (comparison vehicle > AM6545; p < 0.01) (n = 10). Data are expressed as mean ± s.e.m. *p < 0.05, **p < 0.01 by Kruskal-Wallis test.

Fig. 5. Acute AM6545 treatment increases central noradrenergic activity.

A Percentage of mean firing rate in the LC after acute vehicle (VEH) or AM6545 (1 mg/kg) administration respect to baseline values (n = 4). B Percentage of extracellular norepinephrine (NE) levels in the hippocampus after acute vehicle (VEH) or AM6545 (1 mg/kg) administration respect to baseline values (n = 5–11). The arrow indicates the time of administration. C Area under the curve for norepinephrine (NE) levels since the time of vehicle (VEH) or AM6545 (1 mg/kg) administration. D Discrimination index values and E total exploration time obtained in the NORT performed at 48 h mice treated with AM6545 (1 mg/kg) or vehicle (VEH) after bilateral intrahippocampal injection of saline or propranolol (1 μg/μl 0.5 μl per side) (n = 5–6). Data are expressed as mean ± s.e.m. For the firing rate at locus coeruleus *p < 0.05 by two-way repeated measures ANOVA test followed by Bonferroni post hoc. For microdialysis **p < 0.01 by two-way repeated measures ANOVA test and Student’s t-test. For NORT **p < 0.01 (treatment effect) ^#p < 0.05 (pre-treatment effect) by two-way ANOVA test followed by Tukey post hoc.