Ferdinand S. Vilim

From Hunger to Satiety: Reconfiguration of a Feeding Network by Aplysia Neuropeptide Y

A shift in motivational state often produces behavioral change, but the underlying mechanisms are poorly understood. In the marine mollusc, Aplysia californica, feeding-induced transition from a hunger to satiation state leads to a slowdown and an eventual termination of feeding. Because the multifunctional feeding network generates both ingestion and the competing response, egestion, it is possible that the transition from a hunger to a satiety state is associated with network reconfiguration that results in production of fewer ingestive and more egestive responses. Chronic electrophysiological recordings in free-feeding Aplysia showed that as the meal progressed, food elicited fewer ingestive responses and simultaneously increased the number of egestive responses. Injections of Aplysia neuropeptide Y (apNPY) reduced food intake and slowed down the rate of ingestion. apNPY was localized to buccal-ganglion afferents originating in the gut-innervating esophageal nerve (EN), a nerve involved both in satiation and in the generation of egestive programs. During EN stimulation, apNPY was released in the feeding circuit. Importantly, stimulation of the cerebral-buccal interneuron-2, a command-like interneuron that is activated by food and normally elicits ingestive responses, elicited egestive responses in the presence of apNPY. This was accompanied by increased activity of the egestion-promoting interneuron B20 and decreased activity in the ingestion-promoting interneuron B40. Thus, apNPYergic reconfiguration of the feeding central pattern generator plays a role in the gradual transition from hunger to satiety states. More generally, changes in the motivational states may involve not only simple network inhibition but may also require network reconfiguration.

Physiology and biochemistry of peptidergic cotransmission in Aplysia

The marine mollusc Aplysia, whose simple nervous system facilitates study of the neural basis of behavior, was used to investigate the role of peptidergic cotransmission in feeding behavior. Several novel modulatory neuropeptides were purified and localized to identified cholinergic motoneurons. Physiological and biochemical studies demonstrated that these peptides are released when the motoneurons fire at frequencies that occur during normal behavior, and that the peptides modify the relationship between muscle contraction amplitude and relaxation rate so as to maintain optimal motor output when the intensity and frequency of feeding behavior change.

Structure, bioactivity, and cellular localization of myomodulin B: A novel Aplysia peptide

Important insights into mechanisms by which neuromuscular activity can be modulated have been gained by the study of experimentally advantageous preparations such as the ARC neuromuscular system of Aplysia. Previous studies have indicated that one source of modulatory input to the ARC muscle is its own two motor neurons, B15 and B16. Both of these neurons synthesize multiple peptide cotransmitters in addition to their primary neurotransmitter acetylcholine (ACh). Peptides present in the ARC motor neurons include SCPA, SCPB, buccalin A and B, and myomodulin A. We have now purified a novel neuropeptide, myomodulin B, which is structurally similar to myomodulin A. Myomodulin B is present in two identified Aplysia neurons that contain myomodulin A; the ARC motor neuron B16 and the abdominal neuron L10. Ratios of myomodulin A to myomodulin B are approximately 6:1 in both cells. Like myomodulin A, myomodulin B potentiates ARC neuromuscular activity; it acts postsynaptically, and increases the size and relaxation rate of muscle contractions elicited either by motor neuron stimulation or by direct application of ACh to the ARC. When myomodulin A is applied to the ARC in high doses (e.g., at about 10(-7) M), it decreases the size of motor neuron-elicited muscle contractions. This inhibitory effect is never seen with myomodulin B. Thus, despite the structural similarity between the two myomodulins, there exists what may be an important difference in their bioactivity.

Feedforward Compensation Mediated by the Central and Peripheral Actions of a Single Neuropeptide Discovered Using Representational Difference Analysis

Compensatory mechanisms are often used to achieve stability by reducing variance, which can be accomplished via negative feedback during homeostatic regulation. In principle, compensation can also be implemented through feedforward mechanisms where a regulator acts to offset the anticipated output variation; however, few such neural mechanisms have been demonstrated. We provide evidence that an Aplysia neuropeptide, identified using an enhanced representational difference analysis procedure, implements feedforward compensation within the feeding network. We named the novel peptide “allatotropin-related peptide” (ATRP) because of its similarity to insect allatotropin. Mass spectrometry confirmed the peptides identity, and in situ hybridization and immunostaining mapped its distribution in the Aplysia CNS. ATRP is present in the higher-order cerebral-buccal interneuron (CBI) CBI-4, but not in CBI-2. Previous work showed that CBI-4-elicited motor programs have a shorter protraction duration than those elicited by CBI-2. Here we show that ATRP shortens protraction duration of CBI-2-elicited ingestive programs, suggesting a contribution of ATRP to the parametric differences between CBI-4-evoked and CBI-2-evoked programs. Importantly, because Aplysia muscle contractions are a graded function of motoneuronal activity, one consequence of the shortening of protraction is that it can weaken protraction movements. However, this potential weakening is offset by feedforward compensatory actions exerted by ATRP. Centrally, ATRP increases the activity of protraction motoneurons. Moreover, ATRP is present in peripheral varicosities of protraction motoneurons and enhances peripheral motoneuron-elicited protraction muscle contractions. Therefore, feedforward compensatory mechanisms mediated by ATRP make it possible to generate a faster movement with an amplitude that is not greatly reduced, thereby producing stability.

Cerebrin prohormone processing, distribution and action in Aplysia californica

The isolation, characterization, and bioactivity in the feeding circuitry of a novel neuropeptide in the Aplysia californica central nervous system are reported. The 17‐residue amidated peptide, NGGTADALYNLPDLEKIamide, has been termed cerebrin due to its primary location in the cerebral ganglion. Liquid chromatographic purification guided by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry allowed the isolation of the peptide with purity adequate for Edman sequencing. The cerebrin cDNA has been characterized and encodes an 86 amino acid prohormone that predicts cerebrin and one additional peptide. Mapping using in situ hybridization and immunocytochemistry showed that cerebrin containing neuronal somata are localized almost exclusively in the cerebral ganglion, mostly in the F‐ and C‐clusters. Both immunostaining and mass spectrometry demonstrated the presence of cerebrin in the neurohemal region of the upper labial nerve. In addition, immunoreactive processes were detected in the neuropil of all of the ganglia, including the buccal ganglia, and in some interganglionic connectives, including the cerebral‐buccal connective. This suggests that cerebrin may also function as a local signaling molecule. Cerebrin has a profound effect on the feeding motor pattern elicited by the command‐like neuron CBI‐2, dramatically shortening the duration of the radula protraction in a concentration‐dependent manner, mimicking the motor‐pattern alterations observed in food induced arousal states. These findings suggest that cerebrin may contribute to food‐induced arousal in the animal. Cerebrin‐like immunoreactivity is also present in Lymnaea stagnalis suggesting that cerebrin‐like peptides may be widespread throughout gastropoda.

Distinct mechanisms produce functionally complementary actions of neuropeptides that are structurally related but derived from different precursors

Many bioactive neuropeptides containing RFamide at their C terminus have been described in both invertebrates and vertebrates. To obtain insight into the functional logic of RFamide signaling, we investigate it here in the feeding system of Aplysia. We focus on the expression, localization, and actions of two families of RFamide peptides, the FRFamides and FMRFamide, in the central neuronal circuitry and the peripheral musculature that generate the feeding movements. We describe the cloning of the FRFamide precursor protein and show that the FRFamides and FMRFamide are derived from different precursors. We map the expression of the FRFamide and FMRFamide precursors in the feeding circuitry using in situ hybridization and immunostaining and confirm proteolytic processing of the FRFamide precursor by mass spectrometry. We show that the two precursors are expressed in different populations of sensory neurons in the feeding system. In a representative feeding muscle, we demonstrate the presence of both FRFamides and FMRFamide and their release, probably from the processes of the sensory neurons in the muscle. Both centrally and in the periphery, the FRFamides and FMRFamide act in distinct ways, apparently through distinct mechanisms, and nevertheless, from an overall functional perspective, their actions are complementary. Together, the FRFamides and FMRFamide convert feeding motor programs from ingestive to egestive and depress feeding muscle contractions. We conclude that these structurally related peptides, although derived from different precursors, expressed in different neurons, and acting through different mechanisms, remain related to each other in the functional roles that they play in the system.

Neural Analog of Arousal: Persistent Conditional Activation of a Feeding Modulator by Serotonergic Initiators of Locomotion

We investigated how a neural analog of a form of arousal induced by a mildly noxious stimulus can promote two antagonistic responses, locomotion and feeding. Two pairs of cerebral serotonergic interneurons in Aplysia, CC9 and CC10, were persistently activated by transient noxious stimuli. Direct stimulation of CC9–10 activated locomotor activity that outlasted the stimulation and enhanced subsequent nerve-evoked locomotor programs. Thus, CC9–10 function both as initiators and as modulators of the locomotor network. CC9–10 also interacted with the feeding circuit but in a fundamentally different manner. CC9–10 did not directly trigger feeding activity or activate feeding command or pattern generating interneurons. CC9–10 did, however, elicit slow EPSPs in serotonergic cells that modulate feeding responses, the metacerebral cells (MCCs). CC9–10 persistently enhanced MCC excitability, but did not activate the MCCs directly. Previous work has demonstrated that the MCCs are activated during food ingestion via a sensory neuron C2. Interestingly, we found that CC9–10 stimulation converted subthreshold C2 mediated excitation of the MCC into suprathreshold excitation. Transient noxious stimuli also enhanced MCC excitability, and this was largely mediated by CC9–10. To summarize, CC9–10 exert actions on the feeding network, but their functional effects appear to be conditional on the presence of food-related inputs to the MCCs. A potential advantage of this arrangement is that it may prevent conflicting responses from being directly evoked by noxious stimuli while also facilitating the ability of food-related stimuli to generate feeding responses in the aftermath of noxious stimulation.

Composite Modulatory Feedforward Loop Contributes to the Establishment of a Network State

Feedforward loops (FFLs) are one of many network motifs identified in a variety of complex networks, but their functional role in neural networks is not well understood. We provide evidence that combinatorial actions of multiple modulators may be organized as FFLs to promote a specific network state in the Aplysia feeding motor network. The Aplysia feeding central pattern generator (CPG) receives two distinct inputs-a higher-order interneuron cerebral-buccal interneuron-2 (CBI-2) and the esophageal nerve (EN)-that promote ingestive and egestive motor programs, respectively. EN stimulation elicits a persistent egestive network state, which enables the network to temporarily express egestive programs following a switch of input from the EN to CBI-2. Previous work showed that a modulatory CPG element, B65, is specifically activated by the EN and participates in establishing the egestive state by enhancing activity of egestion-promoting B20 interneurons while suppressing activity and synaptic outputs of ingestion-promoting B40 interneurons. Here a peptidergic contribution is mediated by small cardioactive peptide (SCP). Immunostaining and mass spectrometry show that SCP is present in the EN and is released on EN stimulation. Importantly, SCP directly enhances activity and synaptic outputs of B20 and suppresses activity and synaptic outputs of B40. Moreover, SCP promotes B65 activity. Thus the direct and indirect (through B65) pathways to B20 and B40 from SCPergic neurons constitute two FFLs with one functioning to promote egestive output and the other to suppress ingestive output. This composite FFL consisting of the two combined FFLs appears to be an effective means to co-regulate activity of two competing elements that do not inhibit each other, thereby contributing to establish specific network states.

Cloning, expression and processing of the CP2 neuropeptide precursor of Aplysia

The cDNA sequence encoding the CP2 neuropeptide precursor is identified and encodes a single copy of the neuropeptide that is flanked by appropriate processing sites. The distribution of the CP2 precursor mRNA is described and matches the CP2-like immunoreactivity described previously. Single cell RT-PCR independently confirms the presence of CP2 precursor mRNA in selected neurons. MALDI-TOF MS is used to identify additional peptides derived from the CP2 precursor in neuronal somata and nerves, suggesting that the CP2 precursor may give rise to additional bioactive neuropeptides.

Identification of a new neuropeptide precursor reveals a novel source of extrinsic modulation in the feeding system of aplysia

The Aplysia feeding system is advantageous for investigating the role of neuropeptides in behavioral plasticity. One family of Aplysia neuropeptides is the myomodulins (MMs), originally purified from one of the feeding muscles, the accessory radula closer (ARC). However, two MMs, MMc and MMe, are not encoded on the only known MM gene. Here, we identify MM gene 2 (MMG2), which encodes MMc and MMe and four new neuropeptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to verify that these novel MMG2-derived peptides (MMG2-DPs), as well as MMc and MMe, are synthesized from the precursor. Using antibodies against the MMG2-DPs, we demonstrate that neuronal processes that stain for MMG2-DPs are found in the buccal ganglion, which contains the feeding network, and in the buccal musculature including the ARC muscle. Surprisingly, however, no immunostaining is observed in buccal neurons including the ARC motoneurons. In situ hybridization reveals only few MMG2-expressing neurons that are mostly located in the pedal ganglion. Using immunohistochemical and electrophysiological techniques, we demonstrate that some of these pedal neurons project to the buccal ganglion and are the likely source of the MMG2-DP innervation of the feeding network and musculature. We show that the MMG2-DPs are bioactive both centrally and peripherally: they bias egestive feeding programs toward ingestive ones, and they modulate ARC muscle contractions. The multiple actions of the MMG2-DPs suggest that these peptides play a broad role in behavioral plasticity and that the pedal-buccal projection neurons that express them are a novel source of extrinsic modulation of the feeding system of Aplysia.