It has been proposed that a choice of specific behaviors can be mediated either by activation of behavior-specific higher order neurons or by distinct combinations of such neurons in different behaviors. We examined the role that two higher order neurons, CBI-2 and CBI-3, play in the selection of motor programs that correspond to ingestion and egestion, two stimulus-dependent behaviors that are generated by a single central pattern generator (CPG) of Aplysia. We found that CBI-2 could evoke either ingestive, egestive, or ambiguous motor programs depending on the regime of stimulation. When CBI-2 recruited CBI-3 firing via electrical coupling, the motor program tended to be ingestive. In the absence of CBI-3 activation, the program was usually egestive. When CBI-2 was stimulated to produce ingestive programs, hyperpolarization of CBI-3 converted the programs to egestive or ambiguous. When CBI-2 was stimulated to produce egestive or ambiguous programs, co-stimulation of CBI-3 converted them into ingestive. These findings are consistent with the idea that combinatorial commands are responsible for the choice of specific behaviors. Additional support for this view comes from the observations that appropriate stimulus conditions exist both for activation of CBI-2 together with CBI-3, and for activation of CBI-2 without a concomitant activation of CBI-3. The ability of CBI-3 to convert egestive and ambiguous programs into ingestive ones was mimicked by application of APGWamide, a neuropeptide that we have detected in CBI-3 by immunostaining. Thus combinatorial actions of higher order neurons that underlie pattern selection may involve the use of modulators released by specific higher order neurons.

The Aplysia multifunctional feeding central pattern generator (CPG) produces at least two types of motor programs, ingestion and egestion, that involve two sets of radula movements, protraction-retraction and opening-closing movements. In ingestion, the radula closes during retraction to pull food in, whereas in egestion, the radula closes during protraction to push inedible objects out. Thus, radula closure shifts the phase in which it occurs with respect to protraction-retraction in the two programs. To identify the central switching mechanisms, we compared activity of CPG neurons during the two types of motor programs elicited by a higher-order interneuron, cerebral-buccal interneuron-2 (CBI-2). Although CPG elements (B63, B34, and B64) that mediate the protraction-retraction sequence are active in both programs, two other CPG elements, B20 and B4/5, are preferentially active in egestive programs and play a major role in mediating CBI-2-elicited egestive programs. Both B20 and B4/5 control the phasing of radula closure motoneurons (B8 and B16) to ensure that, in egestive programs, these motoneurons fire and produce radula-closing movements only during protraction. Elsewhere, another higher-order interneuron, CBI-3, was shown to convert CBI-2-elicited egestion to ingestion. We show that CBI-3 switches the programs by suppressing the activity of B20 and B4/5. CBI-3, active only during protraction, accomplishes this through fast inhibition of B20 during protraction and slow inhibition of B4/5 during retraction. The slow inhibition is mimicked and occluded by APGWamide, a neuropeptide contained in CBI-3. Thus, fast conventional and slow peptidergic transmissions originating from the same interneuron act in concert to meet specific temporal requirements in pattern switching.

The notion of the command cell has been highly influential in invertebrate neurobiology, and related notions have been increasingly used in research on the vertebrate nervous system. The term “command neuron” implies that the neuron has some critical function in the generation of a normally occurring behavior. Nevertheless, most authors either explicitly or implicitly use a strictly operational definition, independent of considerations of normal behavioral function. That is, command neurons are often defined as neurons which, when stimulated by the experimenter, evoke some behavioral response. Even when utilizing such an operational definition, investigators frequently differ on what they consider to be the exact characteristics that a neuron must have (or not have) to be considered a command cell. A few authors appear to treat command neurons in relation to normal function, but a precise behaviorally relevant definition has not been specified. Because of the ambiguity in the definition of command neurons, the term can refer to a wide variety of neurons which may play divergent behavioral roles. In some ways the attempt to label a cell as a command neuron may interfere with the process of discovering the complex causal determinants of behavior. Nevertheless, the notion that individual cells are responsible for certain behaviors is highly appealing, and an attempt to define the command neuron rigorously could be worthwhile. We suggest that a command neuron be defined as a neuron that is both necessary and sufficient for the initiation of a given behavior. These criteria can by tested by: (1) establishing the response pattern of the putative command neuron during presentation of a given stimulus and execution of a well specified behavior; (2) removing the neuron and showing that the response is no longer elicited by the stimulus (necessary condition); and (3) firing the neuron in its normal pattern and showing that the complete behavioral response occurs (sufficient condition). In some cases, groups of neurons, when treated as a whole, may satisfy the necessity and sufficiency criteria for a given behavior, even though individual neurons of the group fail to meet the criteria. We suggest that such a group be termed a “command system” for the behavior in question. Individual neurons in the command system can be termed “command elements” if, when fired in their normally occurring pattern, they elicit a part of the behavior, or “modulatory elements” if they do not in isolation elicit any response, but alter the behavior produced by other elements in the command system.

Selection of behavioral responses to external stimuli is strongly influenced by internal states, such as intentions and expectations. These internal states are often attributed to higher-order brain functions. Yet here we show that even in the simple feeding network of Aplysia, external stimuli do not directly specify which motor output is expressed; instead, the motor output is specified by the state of the network at the moment of stimulation. The history-dependence of this network state manifests itself in the same way as do intentions and expectations in the behavior of higher animals. Remarkably, we find that activity-dependent plasticity of a synapse within the network itself, rather than some higher-order network, mediates one important aspect of the change in the network state. Through this mechanism, changes in the network state become an automatic consequence of the generation of behavior. Altogether, our findings suggest that intentions and expectations may emerge within behavior-generating networks themselves from the plasticity of the very processes that generate the behavior.A nimal behavior is not merely a passive response to external stimuli; rather, it expresses also the internal state of the animal. This internal state is presumably somehow embodied in the state of the nervous system. Here we study the manifestations and the neurophysiological basis of the internal state in the experimentally advantageous feeding network of the mollusk Aplysia.State-dependence of network function is not a new concept. In the neurophysiological literature, state-dependence is typically discussed as the ability of contextual cues to modify the response to a stimulus. This type of state-dependence has been demonstrated, for example, for locomotion by using stimulation of the mesencephalic locomotor region to elicit walking in the decerebrate cat. The speed of locomotion is determined by the speed of the treadmill on which the cat is placed (1). In another compelling example, stimulation of a command-like neuron in a leech immersed in water elicits swimming, whereas stimulation of the same neuron when the leech is placed on solid substrate elicits crawling (2). Similarly, stimulation of a command-like neuron in a cricket suspended in air elicits avoidance responses but it fails to do so when the cricket is placed on the ground (3). State-dependence of this type is also seen with neuromodulation. For instance, the ability of sensory stimulation to elicit stridulation in the grasshopper is critically dependent on the presence of a muscarinic agonist (4). The setting of network state by application of neuromodulators is a common phenomenon that has been well characterized in simple neuronal networks, such as the stomatogastric system of crustaceans (5-9).This type of state-dependence fails, however, to account for a fundamental feature of the state-dependence that is observed in animal behavior and human psychology. In the type of statedependence just discussed, the behavior is still unambiguously specified by the ext...

Changes in Aplysia biting responses during food arousal are partially mediated by the serotonergic metacerebral cells (MCCs) The nervous system of the marine mollusc Aplysia provides an advantageous model system for the study of the neural basis ofbehavior and the modification ofbehavior by learning and motivational states such as arousal. We have been studying a form of arousal that is elicited by food and is characterized by general changes in locomotion (1) and cardiovascular responses (2) as well as by specific alterations in feeding behavior-such as progressive increases in the strength and speed of biting (1,3,4).Previous studies (5-7) have demonstrated that changes in biting during food arousal are partially mediated by the serotonergic metacerebral cells (MCCs). The MCCs exert central actions within the nervous system and peripheral actions on buccal muscles. We have studied the peripheral actions of the MCCs on the accessory radula closer muscle (ARCM), a muscle used in biting, which is innervated by the MCC and two cholinergic buccal motor neurons B15 and B16 (8). The MCC is not a motor neuron; its activity does not produce ARCM contractions. Instead it increases the strength of contractions produced by stimulation of neurons B15 and B16, presumably by increasing cAMP levels in the muscle (9).This modulatory input from the MCCs, however, accounts for only a part of the manifestations of arousal, because animals in which the MCCs have been lesioned still exhibit a progressive buildup ofthe magnitude and frequency ofbiting, although to a lesser degree (7). This suggests that arousal may be produced by more than one modulatory system. In fact, we have demonstrated that nerve fibers and varicosities in the ARCM contain the neuropeptide SCPB and that exogenous application of SCPB exerts modulatory actions similar to those of serotonin and the MCC-i.e., SCPB increases cAMP in the ARCM and enhances contractions produced by motor neuron stimulation without itself producing a contraction (10). Therefore, there may be peptidergic as well as serotonergic modulation of the ARCM. As a first step in understanding the functional significance of this dual modulation, we have now identified sources of peptidergic input to the ARCM. METHODS Extraction of Peptides from Motor Neurons. Identified neurons B15 and B16 (8) were marked by iontophoresis of fast green dye. In some experiments, peptides were radiolabeled in vivo (11). Buccal ganglia were incubated with 0.5 mCi of [35S]methionine (1 Ci = 37 GBq; Amersham) for 24 hr at 18°C in 1 ml of 50% artificial sea water (ASW)/50% sterile (0.2-,um filtered) hemolymph/100 ,l of antibiotics (penicillin and streptomycin each at 50 units per ml)/colchicine (2.5 ,ul of 1 M colchicine dissolved in Me2SO). Colchicine was added to inhibit axonal transport, which raised intrasomatic peptide levels and reduced, or eliminated, labeled peptides that might be present in fibers and terminals near the somata of dissected neurons (12). After 24 hr, ganglia were rinsed and incubated i...

Coordination of two sets of movements, protraction-retraction versus opening-closing, of the feeding apparatus (the radula) in ingestive and egestive motor programs of Aplysia resembles vertebrate intralimb coordination in that the relative timing of the two sets of movements differs in the two motor programs. In both ingestion and egestion, radula protraction and retraction alternate, whereas radula closure shifts its phase relative to protraction-retraction. In egestion, the radula closes in protraction; in ingestion, the radula closes in retraction. In both ingestive and egestive motor programs elicited by the command-like neuron, cerebral-buccal interneuron-2 (CBI-2), the protraction and retraction movements are mediated by the same sets of controller interneurons. In contrast, radula closure is mediated by two controller interneurons, B20 and B40, that are preferentially active in egestion and ingestion, respectively. In egestion, B20, active in protraction, drives closure motorneuron B8 in protraction, whereas in ingestion, B40, also active in protraction, uses a functionally novel mechanism, fast inhibition and slow excitation, to drive B8 in retraction. Our findings are summarized in a neural model that permits a conceptual comparison of our model with two previous hypothetical models of intralimb coordination in spinal circuits that were proposed by Grillner (1981, 1985) and Berkowitz and Stein (1994). Although our model supports the existence of separate controllers for different movements as in the Grillner (1981, 1985) model; in terms of basic mechanisms, our model is similar to the Berkowitz and Stein (1994) model because the closure movement is mediated by separate controllers in different programs, and thus both models can be classified as recruitment models.

Growing evidence suggests that different forms of complex motor acts are constructed through flexible combinations of a small number of modules in interneuronal networks. It remains to be established, however, whether a module simply controls groups of muscles and functions as a computational unit for use in multiple behaviors (behavior independent) or whether a module controls multiple salient features that define one behavior and is used primarily for that behavior (behavior specific). We used the Aplysia feeding motor network to examine the two proposals by studying the functions of identifiable interneurons. We identified three types of motor programs that resemble three types of behaviors that Aplysia produce: biting, swallowing, and rejection. Two ingestive programs (biting, swallowing) are defined by two movement parameters of the feeding apparatus (the radula): one is the same in both programs (phasing of radula closure motoneurons relative to radula protraction-retraction), whereas the other parameter (protraction duration) is different in the two programs. In each program, these two parameters were specified together by an individual neuron, but the neurons in each were different (B40 for biting, B30 for swallowing). These findings support the existence of behavior-specific modules. Furthermore, neuron B51 was found to mediate a phase that can be flexibly added on to both ingestive and egestive-rejection programs, suggesting that B51 may be a behavior-independent module. The functional interpretation of the role played by these modules is supported by the patterns of synaptic connectivity that they make. Thus, both behavior-specific and behavior-independent modules are used to construct complex behaviors.

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