Romuald Nargeot

In Vitro Analog of Operant Conditioning in Aplysia. II. Modifications of the Functional Dynamics of an Identified Neuron Contribute to Motor Pattern Selection

Previously, an analog of operant conditioning was developed using the buccal ganglia of Aplysia, the probabilistic occurrences of a specific motor pattern (i.e., pattern I), a contingent reinforcement (i.e., stimulation of the esophageal nerve), and monotonic stimulation of a peripheral nerve (i.e., n.2,3). This analog expressed a key feature of operant conditioning (i.e., selective enhancement of the probability of occurrence of a designated motor pattern by contingent reinforcement). In addition, the training induced changes in the dynamical properties of neuron B51, an element of the buccal central pattern generator. To gain insights into the neuronal mechanisms that mediate features of operant conditioning, the present study identified a neuronal element that was critically involved in the selective enhancement of pattern I. We found that bursting activity in cell B51 contributed significantly to the expression of pattern I and that changes in the dynamical properties of this cell were associated with the selective enhancement of pattern I. These changes could be induced by an explicit association of reinforcement with random depolarization of B51. No stimulation of n.2,3 was required. These results indicate that the selection of a designated motor pattern by contingent reinforcement and the underlying neuronal plasticity resulted from the association of reinforcement with a component of central neuronal activity that contributes to a specific motor pattern. The sensory stimulus that allows for occurrences of different motor acts may not be critical for induction of plasticity that mediates the selection of a motor output by contingent reinforcement in operant conditioning.

Behavioral and In Vitro Correlates of Compulsive-Like Food Seeking Induced by Operant Conditioning in Aplysia

Motivated behaviors comprise appetitive actions whose occurrence results partly from an internally driven incentive to act. Such impulsive behavior can also be regulated by external rewarding stimuli that, through learning processes, can lead to accelerated and seemingly automatic, compulsive-like recurrences of the rewarded act. Here, we explored such behavioral plasticity in Aplysia by analyzing how appetitive reward stimulation in a form of operant conditioning can modify a goal-directed component of the animals food-seeking behavior. In naive animals, protraction/retraction cycles of the tongue-like radula are expressed sporadically with highly variable interbite intervals. In contrast, animals that were previously given a food-reward stimulus in association with each spontaneous radula bite now expressed movement cycles with an elevated frequency and a stereotyped rhythmic organization. This rate increase and regularization, which was retained for several hours after training, depended on both the reward quality and its contingency because accelerated, stereotyped biting was not induced in animals that had previously received a less-palatable food stimulus or had been subjected to nonassociative reward stimulation. Neuronal correlates of these learning-induced changes were also expressed in the radula motor pattern-generating circuitry of isolated buccal ganglia. In such in vitro preparations, moreover, manipulation of the burst frequency of the bilateral motor pattern-initiating B63 interneurons indicated that the regularization of radula motor pattern generation in contingently trained animals occurred separately from an increase in cycle rate, thereby suggesting independent processes of network plasticity. These data therefore suggest that operant conditioning can induce compulsive-like actions in Aplysia feeding behavior and provide a substrate for a cellular analysis of the underlying mechanisms.

Functional Organization and Adaptability of a Decision-Making Network in Aplysia

Whereas major insights into the neuronal basis of adaptive behavior have been gained from the study of automatic behaviors, including reflexive and rhythmic motor acts, the neural substrates for goal-directed behaviors in which decision-making about action selection and initiation are crucial, remain poorly understood. However, the mollusk Aplysia is proving to be increasingly relevant to redressing this issue. The functional properties of the central circuits that govern this animal’s goal-directed feeding behavior and particularly the neural processes underlying the selection and initiation of specific feeding actions are becoming understood. In addition to relying on the intrinsic operation of central networks, goal-directed behaviors depend on external sensory inputs that through associative learning are able to shape decision-making strategies. Here, we will review recent findings on the functional design of the central network that generates Aplysia’s feeding-related movements and the sensory-derived plasticity that through learning can modify the selection and initiation of appropriate action. The animal’s feeding behavior and the implications of decision-making will be briefly described. The functional design of the underlying buccal network will then be used to illustrate how cellular diversity and the coordination of neuronal burst activity provide substrates for decision-making. The contribution of specific synaptic and neuronal membrane properties within the buccal circuit will also be discussed in terms of their role in motor pattern selection and initiation. The ability of learning to “rigidify” these synaptic and cellular properties so as to regularize network operation and lead to the expression of stereotyped rhythmic behavior will then be described. Finally, these aspects will be drawn into a conceptual framework of how Aplysia’s goal-directed circuitry compares to the central pattern generating networks for invertebrate rhythmic behaviors.

Cellular and Network Mechanisms of Operant Learning-Induced Compulsive Behavior in Aplysia

BACKGROUND Learning in exploratory and goal-directed behaviors can modify decision-making processes in the initiation of appropriate action and thereby transform the irregular and infrequent expression of such behaviors into inflexible, compulsive-like repetitive actions. However, the neuronal mechanisms underlying such learning-derived behavioral plasticity remain poorly understood. RESULTS Appetitive operant conditioning, a form of associative learning, produces a long-lasting switch in the mollusk Aplysias food-seeking behavior from irregular, impulsive-like radula biting movements into stereotyped, compulsive-like recurrences of this cyclic act. Using isolated buccal ganglia, we recorded intracellularly from an electrically coupled subset of feeding-network neurons whose spontaneous burst discharge is responsible for instigating the motor pattern underlying each radula bite cycle. We report that the sporadic production of biting patterns in preparations from naive and noncontingently trained animals derives from the inherently variable and incoherent bursting of these pattern-initiating neurons that are each randomly capable of triggering a given bite. However, the accelerated rhythmically recurring expression of radula motor patterns after contingent-reward training in vivo arises from a regularization and synchronization of burst discharge in the pattern-initiating cells through a promotion of stereotyped burst-generating oscillations and an increase in the strength of their electrical coupling. CONCLUSIONS Our results show that plasticity in the spatiotemporal organization of pacemaker bursting, both within and between components of an action-initiating neuronal subcircuit, provides novel cellular substrates by which operant learning alters the recurrent expression of a simple goal-directed behavior.

Functional magnetic resonance microscopy at single-cell resolution in Aplysia californica

Significance The direct observation with MRI of neuronal activity at single-neuron resolution represents a significant advancement in magnetic resonance (MR) microscopy and functional neuroimaging. The vast majority of high-resolution MR microscopy studies remain restricted to the generation of static images. This study shifts the focus of MR microscopy from noninvasive static imaging to dynamic investigations of activity in single cells and neuronal networks. Although most functional neuroimaging investigations are limited to averaging the signal from clusters of hundreds of neurons, we show that we are able to record the activity coming from individual neurons and their responses to sensory stimuli. In this work, we show the feasibility of performing functional MRI studies with single-cell resolution. At ultrahigh magnetic field, manganese-enhanced magnetic resonance microscopy allows the identification of most motor neurons in the buccal network of Aplysia at low, nontoxic Mn2+ concentrations. We establish that Mn2+ accumulates intracellularly on injection into the living Aplysia and that its concentration increases when the animals are presented with a sensory stimulus. We also show that we can distinguish between neuronal activities elicited by different types of stimuli. This method opens up a new avenue into probing the functional organization and plasticity of neuronal networks involved in goal-directed behaviors with single-cell resolution.

Neural mechanisms of operant conditioning and learning-induced behavioral plasticity in Aplysia

Associative learning in goal-directed behaviors, in contrast to reflexive behaviors, can alter processes of decision-making in the selection of appropriate action and its initiation, thereby enabling animals, including humans, to gain a predictive understanding of their external environment. In the mollusc Aplysia, recent studies on appetitive operant conditioning in which the animal learns about the positive consequences of its behavior have provided insights into this form of associative learning which, although ubiquitous, remains mechanistically poorly understood. The findings support increasing evidence that central circuit- and cell-wide sites other than chemical synaptic connections, including electrical coupling and membrane conductances controlling intrinsic neuronal excitability and underlying voltage-dependent plateauing or oscillatory mechanisms, may serve as the neural substrates for behavioral plasticity resulting from operant conditioning. Aplysia therefore continues to provide a model system for understanding learning and memory formation that enables establishing the neurobiological links between behavioral, network, and cellular levels of analysis.

Differential Roles of Nonsynaptic and Synaptic Plasticity in Operant Reward Learning-Induced Compulsive Behavior

BACKGROUND Rewarding stimuli in associative learning can transform the irregularly and infrequently generated motor patterns underlying motivated behaviors into output for accelerated and stereotyped repetitive action. This transition to compulsive behavioral expression is associated with modified synaptic and membrane properties of central neurons, but establishing the causal relationships between cellular plasticity and motor adaptation has remained a challenge. RESULTS We found previously that changes in the intrinsic excitability and electrical synapses of identified neurons in Aplysias central pattern-generating network for feeding are correlated with a switch to compulsive-like motor output expression induced by in vivo operant conditioning. Here, we used specific computer-simulated ionic currents in vitro to selectively replicate or suppress the membrane and synaptic plasticity resulting from this learning. In naive in vitro preparations, such experimental manipulation of neuronal membrane properties alone increased the frequency but not the regularity of feeding motor output found in preparations from operantly trained animals. On the other hand, changes in synaptic strength alone switched the regularity but not the frequency of feeding output from naive to trained states. However, simultaneously imposed changes in both membrane and synaptic properties reproduced both major aspects of the motor plasticity. Conversely, in preparations from trained animals, experimental suppression of the membrane and synaptic plasticity abolished the increase in frequency and regularity of the learned motor output expression. CONCLUSIONS These data establish direct causality for the contributions of distinct synaptic and nonsynaptic adaptive processes to complementary facets of a compulsive behavior resulting from operant reward learning.

Implication of dopaminergic modulation in operant reward learning and the induction of compulsive-like feeding behavior in Aplysia

Feeding in Aplysia provides an amenable model system for analyzing the neuronal substrates of motivated behavior and its adaptability by associative reward learning and neuromodulation. Among such learning processes, appetitive operant conditioning that leads to a compulsive-like expression of feeding actions is known to be associated with changes in the membrane properties and electrical coupling of essential action-initiating B63 neurons in the buccal central pattern generator (CPG). Moreover, the food-reward signal for this learning is conveyed in the esophageal nerve (En), an input nerve rich in dopamine-containing fibers. Here, to investigate whether dopamine (DA) is involved in this learning-induced plasticity, we used an in vitro analog of operant conditioning in which electrical stimulation of En substituted the contingent reinforcement of biting movements in vivo. Our data indicate that contingent En stimulation does, indeed, replicate the operant learning-induced changes in CPG output and the underlying membrane and synaptic properties of B63. Significantly, moreover, this network and cellular plasticity was blocked when the input nerve was stimulated in the presence of the DA receptor antagonist cis-flupenthixol. These results therefore suggest that En-derived dopaminergic modulation of CPG circuitry contributes to the operant reward-dependent emergence of a compulsive-like expression of Aplysias feeding behavior.

Correlation between activity in neuron B52 and two features of fictive feeding in Aplysia

The present study examined the correlation between the level of activity neuron B52 and the transition from protraction to retraction phases of buccal motor patterns (BMPs) and the termination of the BMPs. The level of activity in B52 during the protraction phase was positively correlated with the duration of that phase. A second burst of activity in B52 was associated with the termination of the retraction phase. An apparent monosynaptic inhibitory connection from B52 to B64, may mediate the effects of B52. The first burst of activity in B52 delays the onset of activity in B64, thereby prolonging the protraction phase, and the second burst inhibits activity in B64, thereby terminating the retraction phase. These results suggest that activity in B52 may contribute to switching between ingestion-like and rejection-like BMPs by regulating both phase transition and termination of BMPs.

SENSORY-INDUCED PLASTICITY OF MOTOR PATTERN SELECTION IN THE LOBSTER STOMATOGASTRIC NERVOUS SYSTEM

In a previous study, a bilateral sensory input pathway to the crustacean stomatogastric nervous system was reported to induce the functional switching of an identified motor neuron (VD) from one rhythm generating neural network (the pyloric circuit) to another (the cardiac sac network). In the present in vitro study on the spiny lobster, Palinurus vulgaris, we have shown that under certain conditions, repetitive trains of phasic stimulation (1 s; 40 Hz) of one of these sensory nerves elicits either an increase or a decrease in efficacy of the VD switching response. In preparations showing no previous sign either of increase or decrease in VD switching, either response can be induced by prior conditioning stimulation. The increasing effect can be induced by unpaired conditioning stimulation of the contralateral sensory nerve. Conversely, the decrease in switching efficacy is obtained by pairing stimulation of the sensory‐motor pathway with that applied to its contralateral partner. Both the experimentally induced increase and decrease in VD switching are long‐lasting, remaining observable for at least 20 min and in some cases up to 3 h after the original conditioning procedure. Our results suggest that this system provides a suitable ‘simple’ model for the analysis of experience‐related plasticity of the switching of a neuron from one network to another.