Genetic methods for neuronal silencing have great promise for allowing selective inactivation of specific cell types within complex neural systems. Present methods, however, are limited in their reversibility by the slow time scale (days) of transcriptional regulation. We report the rapid and reversible inactivation of mammalian cortical neurons expressing the insect G-protein-coupled receptor AlstR (Drosophila allatostatin receptor) [corrected] after application of its peptide ligand allatostatin (AL). The onset and reversal of inactivation could be achieved rapidly, within minutes. Moreover, the effects of AL were selective for AlstR-transfected neurons. The AlstR/AL system is therefore a promising genetic method for selective and quickly reversible silencing of neuronal activity.

The feeding behavior of Aplysia californica can be classically conditioned using tactile stimulation of the lips as a conditioned stimulus (CS) and food as an unconditioned stimulus (US). Moreover, several neural correlates of classical conditioning have been identified. The present study extended previous work by developing an in vitro analog of classical conditioning and by investigating pairing-specific changes in neuronal and synaptic properties. The preparation consisted of the isolated cerebral and buccal ganglia. Electrical stimulation of a lip nerve (AT 4 ) and a branch of the esophageal nerve (En 2 ) served as the CS and US, respectively. Three protocols were used: paired, unpaired, and US alone. Only the paired protocol produced a significant increase in CS-evoked fictive feeding. At the cellular level, classical conditioning enhanced the magnitude of the CS-evoked synaptic input to pattern-initiating neuron B31/32. In addition, paired training enhanced both the magnitude of the CS-evoked synaptic input and the CS-evoked spike activity in command-like neuron CBI-2. The in vitro analog of classical conditioning reproduced all of the cellular changes that previously were identified following behavioral conditioning and has led to the identification of several new learning-related neural changes. In addition, the pairing-specific enhancement of the CS response in CBI-2 indicates that some aspects of associative plasticity may occur at the level of the cerebral sensory neurons.

Feeding behavior in Aplysia californica can be classically conditioned using tactile stimulation of the lips as conditional stimulus (CS) and food as unconditional stimulus (US) [ (companion paper)]. Conditioning resulted in an increase in the number of CS-evoked bites that persisted for at least 24 hr after training. In this study, neurophysiological correlates of classical conditioning training were identified and characterized in an in vitro preparation of the cerebral and buccal ganglia. Stimulation of a lip nerve (AT(4)), which mediates mechanosensory information, resulted in a greater number of buccal motor patterns (BMPs) in ganglia isolated from animals that had received paired training than in ganglia from control animals. The majority of the evoked BMPs were classified as ingestion-like patterns. Intracellular recordings from pattern-initiating neuron B31/32 revealed that stimulation of AT(4) evoked greater excitatory input in B31/32 in preparations from animals that had received paired training than from control animals. In contrast, excitatory input to buccal neuron B4/5 in response to stimulation of AT(4) was not significantly increased by paired training. Moreover, correlates of classical conditioning were specific to stimulation of AT(4). The number of spontaneously occurring BMPs and the intrinsic properties of two buccal neurons (B4/5 and B31/32) did not differ between groups. These results suggest that appetitive classical conditioning of feeding resulted in the pairing-specific strengthening of the polysynaptic pathway between afferent fibers and pattern-initiating neurons of the buccal central pattern generator.

A training protocol was developed to classically condition feeding behavior in Aplysia californica using tactile stimulation of the lips as the conditional stimulus (CS) and food as the unconditional stimulus (US). Paired training induced a greater increase in the number of bites to the CS than unpaired training or US-only stimulation. Memory for classical conditioning was retained for at least 24 hr. The organization of the reinforcement pathway that supports classical conditioning was analyzed in additional behavioral experiments. No evidence was found for the contribution to appetitive reinforcement of US-mediating pathways originating in the lips of the animals. Bilateral lesions of the anterior branch of the esophageal nerve, which innervates parts of the foregut, however, were found to attenuate classical conditioning. Thus, it appears likely that reinforcement during appetitive classical conditioning of feeding was mediated by afferent pathways that originate in the foregut. The companion paper () describes two neurophysiological correlates of the classical conditioning.

1. Previous computational studies of models of neuron R15 in Aplysia have indicated that several distinct modes of electrical activity may coexist at a given set of parameters, that this multistability can be modulated by transmitters such as serotonin (5-HT) and that brief perturbations of the membrane potential can induce persistent changes in the mode of electrical activity. To test these predictions, the responses of R15 neurons to injections of brief (1.5 s) current pulses were recorded intracellularly in the absence and presence of 5-HT. 2. In the absence of 5-HT, brief perturbations induced abrupt transitions in the electrical activity from bursting to beating. Such transitions were observed in approximately 20% of the cases. The duration of beating activity varied from several seconds to tens of minutes. In the presence of low concentrations (1 microM) of 5-HT, both the probability of mode transitions and the duration of induced beating activity increased significantly. 3. These results indicate that at least two stable modes of electrical activity can coexist in R15 neurons and that this bistability can be regulated by 5-HT. In general, these conclusions agree with the results from analyses of mathematical models of R15. Although the function of these dynamic properties in R15 is speculative, our results, interpreted on the background of the model, support the notion that nonlinear dynamical properties of individual neurons can endow them with richer forms of information processing than has generally been appreciated.