Variable task switching in the feeding network of Aplysia is a function of differential command input.

Command systems integrate sensory information and then activate the interneurons and motor neurons that mediate behavior. Much research has established that the higher-order projection neurons that constitute these systems can play a key role in specifying the nature of the motor activity induced, or determining its parametric features. To a large extent, these insights have been obtained by contrasting activity induced by stimulating one neuron (or set of neurons) to activity induced by stimulating a different neuron (or set of neurons). The focus of our work differs. We study one type of motor program, ingestive feeding in the mollusc Aplysia californica, which can either be triggered when a single projection neuron (CBI-2) is repeatedly stimulated or can be triggered by projection neuron coactivation (e.g., activation of CBI-2 and CBI-3). We ask why this might be an advantageous arrangement. The cellular/molecular mechanisms that configure motor activity are different in the two situations because the released neurotransmitters differ. We focus on an important consequence of this arrangement, the fact that a persistent state can be induced with repeated CBI-2 stimulation that is not necessarily induced by CBI-2/3 coactivation. We show that this difference can have consequences for the ability of the system to switch from one type of activity to another.NEW & NOTEWORTHY We study a type of motor program that can be induced either by stimulating a higher-order projection neuron that induces a persistent state, or by coactivating projection neurons that configure activity but do not produce a state change. We show that when an activity is configured without a state change, it is possible to immediately return to an intermediate state that subsequently can be converted to any type of motor program.

An Anticipatory Circuit Modification That Modifies Subsequent Task Switching.

Modulators are generally expected to establish a network configuration that is appropriate for the current circumstances. We characterize a situation where the opposite is apparently observed. A network effect of a peptide modulator is counterproductive in that it tends to impede rather than promote the creation of the configuration that is appropriate when the modulator is released. This raises a question: why does release occur? We present data that strongly suggest that it impacts task switching. Our experiments were conducted in an Aplysia feeding network that generates egestive and ingestive motor programs. Initial experiments focused on egestive activity and the neuron B8. As activity becomes egestive, there is an increase in synaptic drive to B8 and its firing frequency increases (Wang et al., 2019). We show that, as this occurs, there is also a persistent current that develops in B8 that is outward rather than inward. Dynamic clamp introduction of this current decreases excitability. When there is an egestive-ingestive task switch in Aplysia, negative biasing is observed (i.e., a bout of egestive activity has a negative impact on a subsequent attempt to initiate an ingestive response) (Proekt et al., 2004). Using an in vitro analog of negative biasing, we demonstrate that the outward current that develops during egestive priming plays an important role in establishing this phenomenon. Our data suggest that, although the outward current induced as activity becomes egestive is counterproductive at the time, it plays an anticipatory role in that it subsequently impacts task switching.SIGNIFICANCE STATEMENT In this study, we identify a peptide-induced circuit modification (induction of an outward current) that does not immediately promote the establishment of a behaviorally appropriate network configuration. We ask why this might occur, and present data that strongly suggest that it plays an important role during task switching. Specifically, our data suggest that the outward current we characterize plays a role in the negative biasing that is seen in the mollusc Aplysia when there is a transition from egestive to ingestive activity. It is possible that the mechanism that we describe operates in other species. A negative effect of egestion on subsequent ingestion is observed throughout the animal kingdom.

Convergent effects of neuropeptides on the feeding central pattern generator of Aplysia californica.

These experiments focus on an interneuron (B63) that is part of the feeding central pattern generator (CPG) in Aplysia californica. Previous work has established that B63 is critical for program initiation regardless of the type of evoked activity. B63 receives input from a number of different elements of the feeding circuit. Program initiation occurs reliably when some are activated, but we show that it does not occur reliably with activation of others. When program initiation is reliable, modulatory neuropeptides are released. For example, previous work has established that an ingestive input to the feeding CPG, cerebral buccal interneuron 2 (CBI-2), releases feeding circuit activating peptide (FCAP) and cerebral peptide 2 (CP-2). Afferents with processes in the esophageal nerve (EN) that trigger egestive motor programs release small cardioactive peptide (SCP). Previous studies have described divergent cellular and molecular effects of FCAP/CP-2 and SCP on the feeding circuit that specify motor activity. Here, we show that FCAP/CP-2 and SCP additionally increase the B63 excitability. Thus, we show that peptides that have well-characterized divergent effects on the feeding circuit additionally act convergently at the level of a single neuron. Since convergent effects of FCAP/CP-2 and SCP are not necessary for specifying the type of network output, we ask why they might be important. Our data suggest that they have an impact during a task switch, i.e., when there is a switch from egestive to ingestive activity.NEW & NOTEWORTHY The activity of multifunctional central pattern generators (CPGs) is often configured by neuromodulators that exert divergent effects that are necessary to specify motor output. We demonstrate that ingestive and egestive inputs to the feeding CPG in Aplysia act convergently (as well as divergently). We ask why this convergence may be important and suggest that it may be a mechanism for a type of arousal that occurs during task switching.

Network Degeneracy and the Dynamics of Task Switching in the Feeding Circuit in Aplysia.

The characteristics of a network are determined by parameters that describe the intrinsic properties of the component neurons and their synapses. Degeneracy occurs when more than one set of parameters produces the same (or very similar) output. It is not clear whether network degeneracy impacts network function or is simply a reflection of the fact that, although it is important for a network to be able to generate a particular output, it is not important how this is achieved. We address this issue in the feeding network of the mollusc Aplysia In this system, there are two stimulation paradigms that generate egestive motor programs: repetition priming and positive biasing. We demonstrate that circuit parameters differ in the 2 cases (e.g., egestive repetition priming requires activity in an interneuron, B20, which is not essential for positive biasing). We show that degeneracy has consequences for task switching. If egestive repetition priming is immediately followed by stimulation of an ingestive input to the feeding central pattern generator, the first few cycles of activity are egestive (not ingestive). In this situation, there is a task switch cost. This "cost" is in part due to the potentiating effect of egestive repetition priming on B20. In contrast, there is no switch cost after positive biasing. Stimulation of the ingestive central pattern generator input immediately triggers ingestive activity. Our results indicate that the mechanisms used to pattern activity can impact network function in that they can determine how readily a network can switch from one configuration to another.SIGNIFICANCE STATEMENT A particular pattern of neural activity can be generated by more than one set of circuit parameters. How or whether this impacts network function is unclear. We address this issue in the feeding network of Aplysia and demonstrate that degeneracy in network function can have consequences for task switching. Namely, we show that, when egestive activity is generated via one set of circuit modifications, an immediate switch to ingestive activity is not possible. In contrast, rapid transitions to ingestive activity are possible if egestive activity is generated via a different set of circuit modifications.

Cellular Effects of Repetition Priming in the Aplysia Feeding Network Are Suppressed during a Task-Switch But Persist and Facilitate a Return to the Primed State.

Many neural networks are multitasking and receive modulatory input, which configures activity. As a result, these networks can enter a relatively persistent state in which they are biased to generate one type of output as opposed to another. A question we address is as follows: what happens to this type of state when the network is forced to task-switch? We address this question in the feeding system of the mollusc Aplysia This network generates ingestive and egestive motor programs. We focus on an identified neuron that is selectively active when programs are ingestive. Previous work has established that the increase in firing frequency observed during ingestive programs is at least partially mediated by an excitability increase. Here we identify the underlying cellular mechanism as the induction of a cAMP-dependent inward current. We ask how this current is impacted by the subsequent induction of egestive activity. Interestingly, we demonstrate that this task-switch does not eliminate the inward current but instead activates an outward current. The induction of the outward current obviously reduces the net inward current in the cell. This produces the decrease in excitability and firing frequency required for the task-switch. Importantly, however, the persistence of the inward current is not impacted. It remains present and coexists with the outward current. Consequently, when effects of egestive priming and the outward current dissipate, firing frequency and excitability remain above baseline levels. This presumably has important functional implications in that it will facilitate a return to ingestive activity.SIGNIFICANCE STATEMENT Under physiological conditions, an animal generating a particular type of motor activity can be forced to at least briefly task-switch. In some circumstances, this involves the temporary induction of an "antagonistic" or incompatible motor program. For example, ingestion can be interrupted by a brief period of egestive activity. In this type of situation, it is often desirable for behavioral switching to occur rapidly and efficiently. In this report, we focus on a particular aspect of this type of task-switch. We determine how the priming that occurs when a multitasking network repeatedly generates one type of motor activity can be retained during the execution of an incompatible motor program.

Discovery of leucokinin-like neuropeptides that modulate a specific parameter of feeding motor programs in the molluscan model, Aplysia.

A better understanding of neuromodulation in a behavioral system requires identification of active modulatory transmitters. Here, we used identifiable neurons in a neurobiological model system, the mollusc Aplysia, to study neuropeptides, a diverse class of neuromodulators. We took advantage of two types of feeding neurons, B48 and B1/B2, in the Aplysia buccal ganglion that might contain different neuropeptides. We performed a representational difference analysis (RDA) by subtraction of mRNAs in B48 versus mRNAs in B1/B2. The RDA identified an unusually long (2025 amino acids) peptide precursor encoding Aplysia leucokinin-like peptides (ALKs; e.g. ALK-1 and ALK-2). Northern blot analysis revealed that, compared with other ganglia (e.g. the pedal-pleural ganglion), ALK mRNA is predominantly present in the buccal ganglion, which controls feeding behavior. We then used in situ hybridization and immunohistochemistry to localize ALKs to specific neurons, including B48. MALDI-TOF MS on single buccal neurons revealed expression of 40 ALK precursor-derived peptides. Among these, ALK-1 and ALK-2 are active in the feeding network; they shortened the radula protraction phase of feeding motor programs triggered by a command-like neuron. We also found that this effect may be mediated by the ALK-stimulated enhancement of activity of an interneuron, which has previously been shown to terminate protraction. We conclude that our multipronged approach is effective for determining the structure and defining the diverse functions of leucokinin-like peptides. Notably, the ALK precursor is the first verified nonarthropod precursor for leucokinin-like peptides with a novel, marked modulatory effect on a specific parameter (protraction duration) of feeding motor programs.

Use of the Aplysia feeding network to study repetition priming of an episodic behavior.

Many central pattern generator (CPG)-mediated behaviors are episodic, meaning that they are not continuously ongoing; instead, there are pauses between bouts of activity. This raises an interesting possibility, that the neural networks that mediate these behaviors are not operating under "steady-state" conditions; i.e., there could be dynamic changes in motor activity as it stops and starts. Research in the feeding system of the mollusk Aplysia californica has demonstrated that this can be the case. After a pause, initial food grasping responses are relatively weak. With repetition, however, responses strengthen. In this review we describe experiments that have characterized cellular/molecular mechanisms that produce these changes in motor activity. In particular, we focus on cumulative effects of modulatory neuropeptides. Furthermore, we relate Aplysia research to work in other systems and species, and develop a hypothesis that postulates that changes in response magnitude are a reflection of an efficient feeding strategy.

A clarifying method that improves imaging of Aplysia ganglia.

Improvements in the imaging of neural circuits are essential for studies of network function in both invertebrates and vertebrates. Therefore, CLARITY, a new imaging enhancement technique developed for mouse brains has attracted broad interest from researchers working on other species. We studied the potential of a modified version of CLARITY to enhance the imaging of ganglia in an invertebrate Aplysia. For example, we have modified the hydrogel solution and designed a small container for the Aplysia ganglia. The ganglia were first processed for immunohistochemistry, and then for CLARITY. We examined the compatibility of these techniques and the extent to which the imaging of fluorescence improved using confocal microscopy. We found that CLARITY did indeed enhance the imaging of CP2 immunopositive neurons in Aplysia ganglia. For example, it improved visualization of small, weak immunoreactive neurons deep in the ganglia. Our modifications of CLARITY make this new method suitable for future use in Aplysia experiments. Furthermore, our techniques are likely to facilitate imaging in other invertebrate ganglia.

Specificity of repetition priming: the role of chemical coding.

We investigate stimulus specificity of repetition priming in a tractable model system; the feeding network of Aplysia. Previous studies primarily focused on an aspect of behavior that is altered during ingestive priming, radula opening. Priming of radula opening occurs when two modulatory peptides [feeding circuit activating peptide (FCAP) and cerebral peptide-2 (CP-2)] are released from the cholinergic command-like neuron cerebral buccal interneuron 2. Effects of FCAP/CP-2 on radula opening motor neurons are cAMP mediated. The present experiments sought to determine whether FCAP/CP-2 and cAMP are also involved in the priming of radula opening during an incompatible activity, i.e., during egestive motor programs. Egestive priming is induced when motor programs are triggered by afferents with processes in the esophageal nerve. We demonstrate that egestive priming is not FCAP/CP-2 mediated. Instead, it is induced by an unrelated peptide (small cardioactive peptide), which exerts PKC-mediated effects. Our data, therefore, suggest that different feeding motor programs are primed via actions of different sets of intercellular and intracellular substances. We suggest that this accounts for the stimulus specificity that can be characteristic of repetition priming. Different stimuli activate different central pattern generator inputs. These inputs release different modulators, which induce functionally distinct motor programs.

Functional Characterization of a Vesicular Glutamate Transporter in an Interneuron That Makes Excitatory and Inhibitory Synaptic Connections in a Molluscan Neural Circuit.

Understanding circuit function requires the characterization of component neurons and their neurotransmitters. Previous work on radula protraction in the Aplysia feeding circuit demonstrated that critical neurons initiate feeding via cholinergic excitation. In contrast, it is less clear how retraction is mediated at the interneuronal level. In particular, glutamate involvement was suggested, but was not directly confirmed. Here we study a suspected glutamatergic retraction interneuron, B64. We used the representational difference analysis (RDA) method to successfully clone an Aplysia vesicular glutamate transporter (ApVGLUT) from B64 and from a glutamatergic motor neuron B38. Previously, RDA was used to characterize novel neuropeptides. Here we demonstrate its utility for characterizing other types of molecules. Bioinformatics suggests that ApVGLUT is more closely related to mammalian VGLUTs than to Drosophila and Caenorhabditis elegans VGLUTs. We expressed ApVGLUT in a cell line, and demonstrated that it indeed transports glutamate in an ATP and proton gradient-dependent manner. We mapped the ApVGLUT distribution in the CNS using in situ hybridization and immunocytochemistry. Further, we demonstrated that B64 is ApVGLUT positive, supporting the idea that it is glutamatergic. Although glutamate is primarily an excitatory transmitter in the mammalian CNS, B64 elicits inhibitory PSPs in protraction neurons to terminate protraction and excitatory PSPs in retraction neurons to maintain retraction. Pharmacological data indicated that both types of PSPs are mediated by glutamate. Thus, glutamate mediates the dual function of B64 in Aplysia. More generally, our systematic approaches based on RDA may facilitate analyses of transmitter actions in small circuits with identifiable neurons.

Repetition priming of motor activity mediated by a central pattern generator: the importance of extrinsic vs. intrinsic program initiators.

Repetition priming is characterized by increased performance as a behavior is repeated. Although this phenomenon is ubiquitous, mediating mechanisms are poorly understood. We address this issue in a model system, the feeding network of Aplysia This network generates both ingestive and egestive motor programs. Previous data suggest a chemical coding model: ingestive and egestive inputs to the feeding central pattern generator (CPG) release different modulators, which act via different second messengers to prime motor activity in different ways. The ingestive input to the CPG (neuron CBI-2) releases the peptides feeding circuit activating peptide and cerebral peptide 2, which produce an ingestive pattern of activity. The egestive input to the CPG (the esophageal nerve) releases the peptide small cardioactive peptide. This model is based on research that focused on a single aspect of motor control (radula opening). Here we ask whether repetition priming is observed if activity is triggered with a neuron within the core CPG itself and demonstrate that it is not. Moreover, previous studies demonstrated that effects of modulatory neurotransmitters that induce repetition priming persist. This suggests that it should be possible to "prime" motor programs triggered from within the CPG by first stimulating extrinsic modulatory inputs. We demonstrate that programs triggered after ingestive input activation are ingestive and programs triggered after egestive input activation are egestive. We ask where this priming occurs and demonstrate modifications within the CPG itself. This arrangement is likely to have important consequences for "task" switching, i.e., the cessation of one type of motor activity and the initiation of another.

A d-Amino Acid-Containing Neuropeptide Discovery Funnel.

A receptor binding class of d-amino acid-containing peptides (DAACPs) is formed in animals from an enzymatically mediated post-translational modification of ribosomally translated all-l-amino acid peptides. Although this modification can be required for biological actions, detecting it is challenging because DAACPs have the same mass as their all-l-amino acid counterparts. We developed a suite of mass spectrometry (MS) protocols for the nontargeted discovery of DAACPs and validated their effectiveness using neurons from Aplysia californica. The approach involves the following three steps, with each confirming and refining the hits found in the prior step. The first step is screening for peptides resistant to digestion by aminopeptidase M. The second verifies the presence of a chiral amino acid via acid hydrolysis in deuterium chloride, labeling with Marfey's reagent, and liquid chromatography-mass spectrometry to determine the chirality of each amino acid. The third involves synthesizing the putative DAACPs and comparing them to the endogenous standards. Advantages of the method, the d-amino acid-containing neuropeptide discovery funnel, are that it is capable of detecting the d-form of any common chiral amino acid, and the first two steps do not require peptide standards. Using these protocols, we report that two peptides from the Aplysia achatin-like neuropeptide precursor exist as GdYFD and SdYADSKDEESNAALSDFA. Interestingly, GdYFD was bioactive in the Aplysia feeding and locomotor circuits but SdYADSKDEESNAALSDFA was not. The discovery funnel provides an effective means to characterize DAACPs in the nervous systems of animals in a nontargeted manner.

Complementary interactions between command-like interneurons that function to activate and specify motor programs.

Motor activity is often initiated by a population of command-like interneurons. Command-like interneurons that reliably drive programs have received the most attention, so little is known about how less reliable command-like interneurons may contribute to program generation. We study two electrically coupled interneurons, cerebral-buccal interneuron-2 (CBI-2) and CBI-11, which activate feeding motor programs in the mollusk Aplysia californica. Earlier work indicated that, in rested preparations, CBI-2, a powerful activator of programs, can trigger ingestive and egestive programs. CBI-2 reliably generated ingestive patterns only when it was repeatedly stimulated. The ability of CBI-2 to trigger motor activity has been attributed to the two program-promoting peptides it contains, FCAP and CP2. Here, we show that CBI-11 differs from CBI-2 in that it contains FCAP but not CP2. Furthermore, it is weak in its ability to drive programs. On its own, CBI-11 is therefore less effective as a program activator. When it is successful, however, CBI-11 is an effective specifier of motor activity; that is, it drives mostly ingestive programs. Importantly, we found that CBI-2 and CBI-11 complement each other's actions. First, prestimulation of CBI-2 enhanced the ability of CBI-11 to drive programs. This effect appears to be partly mediated by CP2. Second, coactivation of CBI-11 with CBI-2 makes CBI-2 programs immediately ingestive. This effect may be mediated by specific actions that CBI-11 exerts on pattern-generating interneurons. Therefore, different classes of command-like neurons in a motor network may make distinct, but potentially complementary, contributions as either activators or specifiers of motor activity.

Latent modulation: a basis for non-disruptive promotion of two incompatible behaviors by a single network state.

Behavioral states often preferentially enhance specific classes of behavior and suppress incompatible behaviors. In the nervous system, this may involve upregulation of the efficacy of neural modules that mediate responses to one stimulus and suppression of modules that generate antagonistic or incompatible responses to another stimulus. In Aplysia, prestimulation of egestive inputs [esophageal nerve (EN)] facilitates subsequent EN-elicited egestive responses and weakens ingestive responses to ingestive inputs [Cerebral-Buccal Interneuron (CBI-2)]. However, a single state can also promote incompatible behaviors in response to different stimuli. This is the case in Aplysia, where prestimulation of CBI-2 inputs not only enhances subsequent CBI-2-elicited ingestive responses, but also strengthens EN-elicited egestive responses. We used the modularly organized feeding network of Aplysia to characterize the organizational principles that allow a single network state to promote two opposing behaviors, ingestion and egestion, without the two interfering with each other. We found that the CBI-2 prestimulation-induced state upregulates the excitability of neuron B65 which, as a member of the egestive module, increases the strength of egestive responses. Furthermore, we found that this upregulation is likely mediated by the actions of the neuropeptides FCAP (Feeding Circuit Activating Peptide) and CP2 (Cerebral Peptide 2). This increased excitability is mediated by a form of modulation that we refer to as "latent modulation" because it is established during stimulation of CBI-2, which does not activate B65. However, when B65 is recruited into EN-elicited egestive responses, the effects of the latent modulation are expressed as a higher B65 firing rate and a resultant strengthening of the egestive response.

Functional differentiation of a population of electrically coupled heterogeneous elements in a microcircuit.

Although electrical coupling is present in many microcircuits, the extent to which it will determine neuronal firing patterns and network activity remains poorly understood. This is particularly true when the coupling is present in a population of heterogeneous, or intrinsically distinct, circuit elements. We examine this question in the Aplysia californica feeding motor network in five electrically coupled identified cells, B64, B4/5, B70, B51, and a newly identified interneuron B71. These neurons exhibit distinct activity patterns during the radula retraction phase of motor programs. In a subset of motor programs, retraction can be flexibly extended by adding a phase of network activity (hyper-retraction). This is manifested most prominently as an additional burst in the radula closure motoneuron B8. Two neurons that excite B8 (B51 and B71) and one that inhibits it (B70) are active during hyper-retraction. Consistent with their near synchronous firing, B51 and B71 showed one of the strongest coupling ratios in this group of neurons. Nonetheless, by manipulating their activity, we found that B51 preferentially acted as a driver of B64/B71 activity, whereas B71 played a larger role in driving B8 activity. In contrast, B70 was weakly coupled to other neurons and its inhibition of B8 counteracted the excitatory drive to B8. Finally, the distinct firing patterns of the electrically coupled neurons were fine-tuned by their intrinsic properties and the largely chemical cross-inhibition between some of them. Thus, the small microcircuit of the Aplysia feeding network is advantageous in understanding how a population of electrically coupled heterogeneous neurons may fulfill specific network functions.

Characterization of GdFFD, a D-amino acid-containing neuropeptide that functions as an extrinsic modulator of the Aplysia feeding circuit.

During eukaryotic translation, peptides/proteins are created using L-amino acids. However, a D-amino acid-containing peptide (DAACP) can be produced through post-translational modification via an isomerase enzyme. General approaches to identify novel DAACPs and investigate their function, particularly in specific neural circuits, are lacking. This is primarily due to the difficulty in characterizing this modification and due to the limited information on neural circuits in most species. We describe a multipronged approach to overcome these limitations using the sea slug Aplysia californica. Based on bioinformatics and homology to known DAACPs in the land snail Achatina fulica, we targeted two predicted peptides in Aplysia, GFFD, similar to achatin-I (GdFAD versus GFAD, where dF stands for D-phenylalanine), and YAEFLa, identical to fulyal (YdAEFLa versus YAEFLa), using stereoselective analytical methods, i.e. MALDI MS fragmentation analysis and LC-MS/MS. Although YAEFLa in Aplysia was detected only in an all L-form, we found that both GFFD and GdFFD were present in the Aplysia CNS. In situ hybridization and immunolabeling of GFFD/GdFFD-positive neurons and fibers suggested that GFFD/GdFFD might act as an extrinsic modulator of the feeding circuit. Consistent with this hypothesis, we found that GdFFD induced robust activity in the feeding circuit and elicited egestive motor patterns. In contrast, the peptide consisting of all L-amino acids, GFFD, was not bioactive. Our data indicate that the modification of an L-amino acid-containing neuropeptide to a DAACP is essential for peptide bioactivity in a motor circuit, and thus it provides a functional significance to this modification.

PKC-mediated GABAergic enhancement of dopaminergic responses: implication for short-term potentiation at a dual-transmitter synapse.

Transmitter-mediated homosynaptic potentiation is generally implemented by the same transmitter that mediates the excitatory postsynaptic potentials (EPSPs), e.g., glutamate. When a presynaptic neuron contains more than one transmitter, however, potentiation can in principle be implemented by a transmitter different from that which elicits the EPSPs. Neuron B20 in Aplysia contains both dopamine and GABA. Although only dopamine acts as the fast excitatory transmitter at the B20-to-B8 synapse, GABA increases the size of these dopaminergic EPSPs. We now provide evidence that repeated stimulation of B20 potentiates B20-evoked dopaminergic EPSPs in B8 apparently via a postsynaptic mechanism, and short-term potentiation of this synapse is critical for the establishment and maintenance of an egestive network state. We show that GABA can act postsynaptically to increase dopamine currents that are elicited by direct applications of dopamine to B8 and that dopamine is acting on a 5-HT3-like receptor. This potentiation is mediated by GABAB-like receptors as GABAB-receptor agonists and antagonists, respectively, mimicked and blocked the potentiating actions of GABA. The postsynaptic actions of GABA rely on a G protein-mediated activation of PKC. Our results suggest that the postsynaptic action of cotransmitter-mediated potentiation may contribute to the maintenance of the egestive state of Aplysia feeding network and, in more general terms, may participate in the plasticity of networks that mediate complex behaviors.

Removal of default state-associated inhibition during repetition priming improves response articulation.

Behavior is a product of both the stimuli encountered and the current internal state. At the level of the nervous system, the internal state alters the biophysical properties of, and connections between, neurons establishing a "network state." To establish a network state, the nervous system must be altered from an initial default/resting state, but what remains unclear is the extent to which this process represents induction from a passive default state or the removal of suppression by an active default state. We use repetition priming (a history-dependent improvement of behavioral responses to repeatedly encountered stimuli) to determine the cellular mechanisms underlying the transition from the default to the primed network state. We demonstrate that both removal of active suppression and induction of neuron excitability changes each contribute separately to the production of a primed state. The feeding system of Aplysia californica displays repetition priming via an increase in the activity of the radula closure neuron B8, which results in increased bite strength with each motor program. We found that during priming, B8 received progressively less inhibitory input from the multifunctional neurons B4/5. Additionally, priming enhanced the excitability of B8, but the rate at which B8 activity increased as a result of these changes was regulated by the progressive removal of inhibitory input. Thus, the establishment of the network state involves the induction of processes from a rested state, yet the consequences of these processes are conditional upon critical gating mechanisms actively enforced by the default state.

Release of a single neurotransmitter from an identified interneuron coherently affects motor output on multiple time scales.

Neurotransmitters can have diverse effects that occur over multiple time scales often making the consequences of neurotransmission difficult to predict. To explore the consequences of this diversity, we used the buccal ganglion of Aplysia to examine the effects of GABA release by a single interneuron, B40, on the intrinsic properties and motor output of the radula closure neuron B8. B40 induces a picrotoxin-sensitive fast IPSP lasting milliseconds in B8 and a slow EPSP lasting seconds. We found that the excitatory effects of this slow EPSP are also mediated by GABA. Together, these two GABAergic actions structure B8 firing in a pattern characteristic of ingestive programs. Furthermore, we found that repeated B40 stimulation induces a persistent increase in B8 excitability that was occluded in the presence of the GABA B receptor agonist baclofen, suggesting that GABA affects B8 excitability over multiple time scales. The phasing of B8 activity during the feeding motor programs determines the nature of the behavior elicited during that motor program. The persistent increase in B8 excitability induced by B40 biased the activity of B8 during feeding motor programs causing the motor programs to become more ingestive in nature. Thus, a single transmitter released from a single interneuron can have consequences for motor output that are expressed over multiple time scales. Importantly, despite the differences in their signs and temporal characteristics, the three actions of B40 are coherent in that they promote B8 firing patterns that are characteristic of ingestive motor outputs.

A Single Central Pattern Generator for the Control of a Locomotor Rolling Wave in Mollusc Aplysia.

Locomotion in mollusc Aplysia is implemented by a pedal rolling wave, a type of axial locomotion. Well-studied examples of axial locomotion (pedal waves in Drosophila larvae and body waves in leech, lamprey, and fish) are generated in a segmented nervous system via activation of multiple coupled central pattern generators (CPGs). Pedal waves in molluscs, however, are generated by a single pedal ganglion, and it is unknown whether there are single or multiple CPGs that generate rhythmic activity and phase shifts between different body parts. During locomotion in intact Aplysia, bursting activity in the parapedal commissural nerve (PPCN) was found to occur during tail contraction. A cluster of 20 to 30 P1 root neurons (P1Ns) on the ventral surface of the pedal ganglion, active during the pedal wave, were identified. Computational cluster analysis revealed that there are 2 phases to the motor program: phase I (centered around 168°) and phase II (centered around 357°). PPCN activity occurs during phase II. The majority of P1Ns are motoneurons. Coactive P1Ns tend to be electrically coupled. Two classes of pedal interneurons (PIs) were characterized. Class 1 (PI1 and PI2) is active during phase I. Their axons make a loop within the pedal ganglion and contribute to locomotor pattern generation. They are electrically coupled to P1Ns that fire during phase I. Class 2 (PI3) is active during phase II and innervates the contralateral pedal ganglion. PI3 may contribute to bilateral coordination. Overall, our findings support the idea that Aplysia pedal waves are generated by a single CPG.