Reversible inactivation of the nucleus basalis magnocellularis induces disruption of cortical acetylcholine release and acquisition, but not retrieval, of aversive memories (conditioning taste aversionyfree-moving microdialysisygustatory cortexycholinergic basal forebrain)

The basal forebrain complex, which includes the nucleus basalis magnocellularis (NBM), provides widespread cholinergic and g-aminobutyric acid-containing projections throughout the brain, including the insular and pyriform cortices. A number of studies have implicated the cholinergic neurons in the mediation of learning and memory processes. However, the role of basal forebrain activity in information retrieval mechanisms is less known. The aim of the present study is to evaluate the effects of reversible inactivation of the NBM by tetrodotoxin (TTX, a voltagesensitive sodium channel blocker) during the acquisition and retrieval of conditioned taste aversion (CTA) and to measure acetylcholine (ACh) release during TTX inactivation in the insular cortex, by means of the microdialysis technique in free-moving rats. Bilateral infusion of TTX in the NBM was performed 30 min before the presentation of gustative stimuli, in either the CTA acquisition trial or retrieval trial. At the same time, levels of extracellular ACh release were measured in the insular cortex. The behavioral results showed significant impairment in CTA acquisition when the TTX was infused in the NBM, whereas retrieval was not affected when the treatment was given during the test trial. Biochemical results showed that TTX infusion into the NBM produced a marked decrease in cortical ACh release as compared with the controls during consumption of saccharin in the acquisition trial. Depleted ACh levels were found during the test trial in all groups except in the group that received TTX during acquisition. These results suggest a cholinergic-dependent process during acquisition, but not during memory retrieval, and that NBM-mediated cholinergic cortical release may play an important role in early stages of learning, but not during recall of aversive memories. The cerebral cortex has been considered as the ubiquitous place for the storage of long-term memory. In this regard, a great deal of investigation into brain health has focused on a particular central nervous system disorder, Alzheimer’s disease. This disease results, at least in part, from a deficit in acetylcholine (ACh) neurotransmission caused by a degeneration of large basal forebrain (BF) cholinergic neurons and a deficit in choline acetyltransferase, the enzyme that synthesizes ACh (1). Accumulative evidence supports the role of cholinergic neurons in the BF in processes such as arousal, attention, learning, and memory. Behavioral deficits associated with lesions produced by injections of excitatory amino acid agonists into the nucleus basalis magnocellularis (NBM) have been demonstrated in a variety of tasks (2, 3). However, the behavioral deficits of these lesions might be caused not only by the resulting cholinergic deafferentation, because 30–35% of the population of BF projections neurons to the cortex are g-aminobutyric acid-containing neurons (4–6). In this regard, there are several experiments trying to study more directly the modulatory role of ACh in the activity of cortical neurons, which have brought their observations from electrophysiological and biochemical studies. It has been reported that ACh is released in the rat neocortex in response to a variety of behavioral and environmental conditions, including wakefulness (7), motor activity (8), restraint, or handlinginduced stress (9) and by exposure to auditory, visual, and gustatory stimulation (10–12). In addition to these findings, which demonstrate differences in ACh release related to different states of arousal, other evidence suggests that associative conditioning (13–16) also may modify ACh release in the neocortex. Thus, it has been demonstrated that a significant enhancement of ACh release is associated with appetitive conditioning procedures. Training for tactile discrimination causes a specific enhancement in ACh release in the somatosensory cortex that is related to acquisition of discrimination performance (17). Additionally, Shimura and coworkers (12), using in vivo microdialysis, have shown that ACh release in the insular cortex (IC) is influenced by behavioral expression of aversive taste stimuli. Conditioned taste aversion (CTA) has been widely used for studying the neurobiology of learning processes; the animal acquires aversion to a taste cue when it is followed by digestive malaise. The CTA model contains clearly defined anatomical substrates (18, 19), and several studies have demonstrated that the insular gustatory neocortex is strongly involved in the mnemonic representation of taste and can be disrupted by NBM lesions (20) or cortical cholinergic antagonists (21). Recently, experiments of NBM lesions have brought a differential role of cholinergic activity during learning and memory. For example, bilateral cortical administration of antibodies for nerve growth factor produced a nearly total loss of cortical ACh levels, and a significant decrement of cells in the NBM, as well as impaired acquisition, but not retrieval, of conditioned taste aversion and inhibitory avoidance (22). However, the role of the cortical ACh release, mediated by NBM afferents, during acquisition and retrieval of taste aversion memory, has not been assessed directly. The purpose of the present experiment was to measure the effects of the bilateral blockage of the NBM on the ACh release in the IC of rats during acquisition and retrieval of CTA. To determine the in vivo effects, we used a microdialysis technique for freely moving animals to measure ACh release The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas.org. Abbreviations: NBM, nucleus basalis magnocellularis; TTX, tetrodotoxin; CTA, conditioned taste aversion; ACh, acetylcholine; IC, insular cortex; BF, basal forebrain. *To whom reprint requests should be addressed at: Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México, Apdo. Postal 70–253, 04510, Mexico D.F. e-mail: fbermude@ifisiol. unam.mx.