Abstract—Neurochemical alterations in Alzheimer’s disease (AD) include cholinergic neuronal loss in the nucleus basa- lis of Meynert (nbM) and a decrease in densities of the M2 muscarinic receptor subtype in areas related to learning and memory. Neuromodulators present in the cholinergic pathways, such as neuropeptides and neurolipids, control these cognitive processes and have become targets of research in order to understand and treat the pathophysio- logical and clinical stages of the disease. This is the case of the endocannabinoid and galaninergic systems, which have been found to be up-regulated in AD, and could there- fore have a neuroprotective role. In the present study, the functional coupling of G/o protein-coupled receptors to GalR1, and the CB1 receptor subtype for endocannabinoids were analyzed in the 3xTg-AD mice model of AD. In addition, the activity mediated by Gi/o protein-coupled M2/4 muscarinic receptor subtypes was also analyzed in brain areas involved in anxiety and cognition. Thus, male mice were studied at 4 and 15 months of age (prodromal and advanced stages, respectively) and compared to age-matched non-transgenic (NTg) mice (adult and old, respectively). In 4-month-old 3xTg-AD mice, the [35S]GTPcS binding stimulated by galanin was significantly increased in the hypothalamus, but a decrease of functional M2/4 receptors was observed in the posterior amygdala. The CB1 cannabinoid receptor activity was up-regulated in the ante- rior thalamus at that age. In 15-month-old 3xTg-AD mice, muscarinic receptor activity was found to be increased in motor cortex, while CB1 activity was decreased in nbM. No changes were found in GalR1-mediated activity at this age. Our results provide further evidence of the relevance of lim- bic areas in the prodromal stage of AD, the profile of which is characterized by anxiety. The up-regulation of galaniner- gic and endocannabinoid systems support the hypothesis.