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.

Urotensin II in invertebrates: from structure to function in Aplysia californica.

Neuropeptides are ancient signaling molecules that are involved in many aspects of organism homeostasis and function. Urotensin II (UII), a peptide with a range of hormonal functions, previously has been reported exclusively in vertebrates. Here, we provide the first direct evidence that UII-like peptides are also present in an invertebrate, specifically, the marine mollusk Aplysia californica. The presence of UII in the central nervous system (CNS) of Aplysia implies a more ancient gene lineage than vertebrates. Using representational difference analysis, we identified an mRNA of a protein precursor that encodes a predicted neuropeptide, we named Aplysia urotensin II (apUII), with a sequence and structural similarity to vertebrate UII. With in-situ hybridization and immunohistochemistry, we mapped the expression of apUII mRNA and its prohormone in the CNS and localized apUII-like immunoreactivity to buccal sensory neurons and cerebral A-cluster neurons. Mass spectrometry performed on individual isolated neurons, and tandem mass spectrometry on fractionated peptide extracts, allowed us to define the posttranslational processing of the apUII neuropeptide precursor and confirm the highly conserved cyclic nature of the mature neuropeptide apUII. Electrophysiological analysis of the central effects of a synthetic apUII suggests it plays a role in satiety and/or aversive signaling in feeding behaviors. Finding the homologue of vertebrate UII in the numerically small CNS of an invertebrate animal model is important for gaining insights into the molecular mechanisms and pathways mediating the bioactivity of UII in the higher metazoan.

Coordination of distinct motor structures through remote axonal coupling of projection interneurons.

Complex behaviors often require coordinated movements of dissimilar motor structures. The underlying neural mechanisms are poorly understood. We investigated cycle-by-cycle coordination of two dissimilar feeding structures in Aplysia californica: the external lips and the internal radula. During feeding, the lips open while the radula protracts. Lip and radula motoneurons are located in the cerebral and buccal ganglia, respectively, and radula motoneurons are controlled by a well characterized buccal central pattern generator (CPG). Here, we examined whether the three electrically coupled lip motoneurons C15/16/17 are controlled by the buccal CPG or by a previously postulated cerebral CPG. Two buccal-cerebral projection interneurons, B34 and B63, which are part of the buccal CPG and mediate radula protraction, monosynaptically excite C15/16/17. Recordings from the B34 axon in the cerebral ganglion demonstrate its direct electrical coupling with C15/16/17, eliminating the need for a cerebral CPG. Moreover, when the multifunctional buccal CPG generates multiple forms of motor programs due to the activation of two inputs, the command-like neuron CBI-2 and the esophageal nerve (EN), C15/16 exhibit activity patterns that are distinct from C17. These distinct activity patterns result from combined activity of B34 and B63 and their differential excitation of C15/16 versus C17. In more general terms, we identified neuronal mechanisms that allow a single CPG to coordinate the phasing and activity of remotely located motoneurons innervating distinct structures that participate in the production of different motor outputs. We also demonstrated that axodendritic electrical coupling by projection interneurons plays a pivotal role in coordinating activity of these remotely located neurons.

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.

Distinct inhibitory neurons exert temporally specific control over activity of a motoneuron receiving concurrent excitation and inhibition.

Recent work suggests that concurrent excitation and inhibition originating in central pattern generators (CPGs) may be used to control rhythmic motoneuronal activity. The specific roles that the inhibition plays in such cases are not well understood, however, in part because of the lack of identification of presynaptic inhibitory neurons. Here we demonstrate that, in the Aplysia feeding CPG, inhibitory inputs may be critical for flexible control of the activity of motoneurons in different forms of behavior. The feeding CPG generates ingestive and egestive motor programs, differing in the high and low activity, respectively, of the motoneuron B8 during the retraction phase of the programs. We show that, during retraction, B8 receives concurrent excitation and inhibition that produces a high-conductance state. The inhibition originates in two types of CPG neurons, B4/5 and B70, that are more active in egestion than ingestion and play a role in suppressing B8 activity during egestion. In turn, the activities of both B4/5 and B70 are suppressed by the ingestion-promoting descending interneuron CBI-3 (for cerebral-buccal interneuron 3). Thus, concurrent excitation and inhibition may be an effective means of controlling motoneuronal activity in a behavior-dependent manner. More detailed analyses reveal, furthermore, that B4/5 and B70 exert complementary actions by acting preferentially in the early and late part of retraction, respectively. Thus, the use of multiple neurons to generate inhibitory inputs to motoneurons that receive concurrent excitation and inhibition brings an additional level of flexibility that allows a temporally specific control of motoneuronal activity within a single phase of motor programs.

An input-representing interneuron regulates spike timing and thereby phase switching in a motor network.

Despite the importance of spike-timing regulation in network functioning, little is known about this regulation at the cellular level. In the Aplysia feeding network, we show that interneuron B65 regulates the timing of the spike initiation of phase-switch neurons B64 and cerebral-buccal interneuron-5/6 (CBI-5/6), and thereby determines the identity of the neuron that acts as a protraction terminator. Previous work showed that B64 begins to fire before the end of protraction phase and terminates protraction in CBI-2-elicited ingestive, but not in CBI-2-elicited egestive programs, thus indicating that the spike timing and phase-switching function of B64 depend on the type of the central pattern generator (CPG)-elicited response rather than on the input used to activate the CPG. Here, we find that CBI-5/6 is a protraction terminator in egestive programs elicited by the esophageal nerve (EN), but not by CBI-2, thus indicating that, in contrast to B64, the spike timing and protraction-terminating function of CBI-5/6 depends on the input to the CPG rather than the response type. Interestingly, B65 activity also depends on the input in that B65 is highly active in EN-elicited programs, but not in CBI-2-elicited programs independent of whether the programs are ingestive or egestive. Notably, during EN-elicited egestive programs, hyperpolarization of B65 delays the onset of CBI-5/6 firing, whereas in CBI-2-elicited ingestive programs, B65 stimulation simultaneously advances CBI-5/6 firing and delays B64 firing, thereby substituting CBI-5/6 for B64 as the protraction terminator. Thus, we identified a neural mechanism that, in an input-dependent manner, regulates spike timing and thereby the functional role of specific neurons.

State dependence of spike timing and neuronal function in a motor pattern generating network.

When sustained firing of a neuron is similar in different types of motor programs, its role in the generation of these programs is often similar. We investigated whether this is also the case for neurons involved in phase transition. In the Aplysia feeding central pattern generator (CPG), identified interneuron B64 starts firing at the transition between the protraction and the retraction phases of all types of motor programs, and its firing is sustained during the retraction phase. It was thought that B64 functions as a protraction terminator as it provides strong inhibitory input to protraction interneurons and motoneurons. Furthermore, premature activation of B64 can lead to premature termination of the protraction phase. Indeed, as we show here, B64 can terminate the protraction phase regardless of the type of motor program. However, B64 actually only functions as a protraction terminator in ingestive-like but not in egestive-like programs. This differential role of B64 results from a differential timing of the initiation of B64 spiking in the two types of programs. In turn, this differential timing of the initiation of B64 firing is determined by the internal state of the CPG. Thus, this study indicates the importance of the timing of initiation of firing in determining the functional role of a neuron and demonstrates that this role depends on the activity-dependent state of the network.

Feeding CPG in Aplysia directly controls two distinct outputs of a compartmentalized interneuron that functions as a CPG element.

In the context of motor program generation in Aplysia, we characterize several functional aspects of intraneuronal compartmentalization in an interganglionic interneuron, CBI-5/6. CBI-5/6 was shown previously to have a cerebral compartment (CC) that includes a soma that does not generate full-size action potentials and a buccal compartment (BC) that does. We find that the synaptic connections made by the BC of CBI-5/6 in the buccal ganglion counter the activity of protraction-phase neurons and reinforce the activity of retraction-phase neurons. In buccal motor programs, the BC of CBI-5/6 fires phasically, and its premature activation can phase advance protraction termination and retraction initiation. Thus the BC of CBI-5/6 can act as an element of the central pattern generator (CPG). During protraction, the CC of CBI-5/6 receives direct excitatory inputs from the CPG elements, B34 and B63, and during retraction, it receives antidromically propagating action potentials that originate in the BC of CBI-5/6. Consequently, in its CC, CBI-5/6 receives depolarizing inputs during both protraction and retraction, and these depolarizations can be transmitted via electrical coupling to other neurons. In contrast, in its BC, CBI-5/6 uses spike-dependent synaptic transmission. Thus the CPG directly and differentially controls the program phases in which the two compartments of CBI-5/6 may transmit information to its targets.

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.

Peptidergic innervation of the vasoconstrictor muscle of the abdominal aorta in Aplysia kurodai.

The arterial system of the marine mollusc Aplysia consists of three major arteries. One of them, the abdominal aorta, has a sphincter (the vasoconstrictor muscle) at the base of the artery. Contraction of this muscle reduces the blood flow into the abdominal aorta, thereby, playing a role in the regulation of the blood distribution in Aplysia. Here, we show the contractility of the vasoconstrictor muscle is modulated by three types of endogenous peptides, Aplysia mytilus inhibitory peptide-related peptides (AMRP), enterin and NdWFamide. Immunohistochemistry showed that putative neuronal processes containing the three peptides exist in the vasoconstrictor muscle. Enterin inhibited the muscle contraction elicited by the nerve stimulation or the application of a putative excitatory transmitter, acetylcholine (ACh). Enterin hyperpolarized the resting potential of the muscle and decreased the amplitude of the excitatory junction potential (EJP). AMRP also inhibited the nerve-evoked contraction although its action on the ACh-induced contraction was variable. AMRP also reduced the size of EJP, but had no effect on the resting potential of the muscle. NdWFamide enhanced the nerve-evoked contraction but not the ACh-induced contraction. NdWFamide augmented EJP without affecting the resting potential of the muscle. These results suggest that AMRP, enterin and NdWFamide are endogenous modulators of the contractile activity of the vasoconstrictor muscle, and that the peptidergic innervations of this muscle contribute to fine tuning of the blood distribution in Aplysia.

Distribution of the Aplysia cardioexcitatory peptide, NdWFamide, in the central and peripheral nervous systems of Aplysia.

NdWFamide is an Aplysia cardioexcitatory tri-peptide containing D-tryptophan. To investigate the roles of this peptide, we examined the immunohistochemical distribution of NdWFamide-positive neurons in Aplysia tissues. All the ganglia of the central nervous system (CNS) contained NdWFamide-positive neurons. In particular, two left upper quadrant cells in the abdominal ganglion, and the anterior cells in the pleural ganglion showed extensive positive signals. NdWFamide-positive processes were observed in peripheral tissues, such as those of the cardio-vascular system, digestive tract, and sex-accessory organs, and in the connectives or neuropils in the CNS. NdWFamide-positive neurons were abundant in peripheral plexuses, such as the stomatogastric ring. To examine the NdWFamide contents of tissues, we fractionated peptidic extracts from the respective tissues by reversed-phase high-pressure liquid chromatography and then assayed the fractions by competitive enzyme-linked immunosorbent assay. A fraction corresponding to the retention time of synthetic NdWFamide contained the most immunoreactivity, indicating that the tissues contained NdWFamide. The prevalence of the NdWFamide content was roughly in the order: abdominal ganglion >heart >gill >blood vessels >digestive tract. In most of the tissues containing NdWFamide-positive nerves, NdWFamide modulated the motile activities of the tissues. Thus, NdWFamide seems to be a versatile neurotransmitter/modulator of Aplysia and probably regulates the physiological activities of this animal.

Structural and functional diversities of the Aplysia Mytilus inhibitory peptide-related peptides.

Aplysia Mytilus inhibitory peptide-related peptides (AMRPs) are multiple hexapeptides coded on a single precursor. By comparing the AMRP precursors of two species of Aplysia (Aplysia californica and Aplysia kurodai), we found that there are substantial numbers of species-specific AMRPs. We next compared the function of AMRPs on the anterior aorta between A. kurodai and Aplysia juliana. In A. juliana, AMRPs inhibited the contractile activity of the aorta (EC(50)=10(-9) to 10(-8)M), whereas the peptides had no obvious action in A. kurodai up to 10(-7)M. These results indicate that AMRPs are both structurally and functionally diverse neuropeptides even among closely related species.

The enterins inhibit the contractile activity of the anterior aorta of Aplysia kurodai.

The anterior aorta is one of the largest blood vessels in the marine mollusc Aplysia kurodai. We examined the actions of recently identified neuropeptides, the enterins, on this blood vessel. Immunohistochemistry revealed that the enterin-immunopositive nerve fibers and varicosity-like structures are abundant in the aorta. When the enterins were applied to the aorta, the basal tonus of the arterial muscles was diminished. The enterins also decreased the contraction amplitude of the anterior aorta evoked either by the application of an Aplysia cardioactive peptide, NdWFamide, or by the stimulation of a nerve innervating the aorta (the vulvar nerve). We found that the enterins activate the 4-aminopyridine (4-AP)-sensitive K(+) channels, and thereby hyperpolarize the membrane potential of the aortic muscles. In the presence of 4-AP, the enterins failed to inhibit the muscle contraction evoked by the vulvar nerve stimulation, suggesting that the inhibition is mainly due to the activation of the 4-AP-sensitive K(+) channels. The inhibition of the NdWFamide-evoked contraction by the enterin was not, however, affected by 4-AP. These results suggest that the enterins are involved in inhibitory regulation of the contractile activity of the anterior aorta, and that the inhibition could be due to multiple mechanisms.