The roles of co-transmission in neural network modulation.

Neuromodulation provides considerable flexibility to the output of neural networks. In spite of the extensive literature documenting the presence of modulatory peptide co-transmitters in many neurons, considerably less is known about the specific roles of co-transmission in circuit function. This review describes some of the potential consequences of peptide co-transmission in functional circuits, using specific examples from recent work on the actions of identified peptidergic projection neurons acting on the multifunctional neural network within the crustacean stomatogastric ganglion. This system reveals that co-transmission provides projection neurons with a rich assortment of strategies for eliciting multiple outputs from a multifunctional network.

Species-specific modulation of pattern-generating circuits.

Phylogenetic comparison can reveal general principles governing the organization and neuromodulation of neural networks. Suitable models for such an approach are the pyloric and gastric motor networks of the crustacean stomatogastric ganglion (STG). These networks, which have been well studied in several species, are extensively modulated by projection neurons originating in higher-order ganglia. Several of these have been identified in different decapod species, including the paired modulatory proctolin neuron (MPN) in the crab Cancer borealis [Nusbaum & Marder (1989) J. Neurosci., 9,1501-1599; Nusbaum & Marder (1989), J. Neurosci., 9, 1600-1607] and the apparently equivalent neuron pair, called GABA (gamma-aminobutyric acid) neurons 1 and 2 (GN1/2), in the lobster Homarus gammarus [Cournil et al. (1990) J. Neurocytol., 19, 478-493]. The morphologies of MPN and GN1/2 are similar, and both exhibit GABA-immunolabelling. However, unlike MPN, GN1/2 does not contain the peptide transmitter proctolin. Instead, GN1/2, but not MPN, is immunoreactive for the neuropeptides related to cholecystokinin (CCK) and FLRFamide. Nonetheless, GN1/2 excitation of the lobster pyloric rhythm is similar to the proctolin-mediated excitation of the crab pyloric rhythm by MPN. In contrast, GN1/2 and MPN both use GABA but produce opposite effects on the gastric mill rhythm. While MPN stimulation produces a GABA-mediated suppression of the gastric rhythm [Blitz & Nusbaum (1999) J. Neurosci., 19, 6774-6783], GN1/2 activates or enhances gastric rhythmicity. These results highlight the care needed when generalizing neuronal organization and function across related species. Here we show that the 'same' neuron in different species does not contain the same neurotransmitter complement, nor does it exert all of the same effects on its postsynaptic targets. Conversely, a different transmitter phenotype is not necessarily associated with a qualitative change in the way that a modulatory neuron influences target network activity.

GABA and responses to GABA in the stomatogastric ganglion of the crab Cancer borealis.

The multifunctional neural circuits in the crustacean stomatogastric ganglion (STG) are influenced by many small-molecule transmitters and neuropeptides that are co-localized in identified projection neurons to the STG. We describe the pattern of gamma-aminobutyric acid (GABA) immunoreactivity in the stomatogastric nervous system of the crab Cancer borealis and demonstrate biochemically the presence of authentic GABA in C. borealis. No STG somata show GABA immunoreactivity but, within the stomatogastric nervous system, GABA immunoreactivity co-localizes with several neuropeptides in two identified projection neurons, the modulatory proctolin neuron (MPN) and modulatory commissural neuron 1 (MCN1). To determine which actions of these neurons are evoked by GABA, it is necessary to determine the physiological actions of GABA on STG neurons. We therefore characterized the response of each type of STG neuron to focally applied GABA. All STG neurons responded to GABA. In some neurons, GABA evoked a picrotoxin-sensitive depolarizing, excitatory response with a reversal potential of approximately -40 mV. This response was also activated by muscimol. In many STG neurons, GABA evoked inhibitory responses with both K(+)- and Cl(-)-dependent components. Muscimol and beta-guanidinopropionic acid weakly activated the inhibitory responses, but many other drugs, including bicuculline and phaclofen, that act on vertebrate GABA receptors were not effective. In summary, GABA is found in projection neurons to the crab STG and can evoke both excitatory and inhibitory actions on STG neurons.

Projection neurons with shared cotransmitters elicit different motor patterns from the same neural circuit.

Specificity in the actions of different modulatory neurons is often attributed to their having distinct cotransmitter complements. We are assessing the validity of this hypothesis with the stomatogastric nervous system of the crab Cancer borealis. In this nervous system, the stomatogastric ganglion (STG) contains a multifunctional network that generates the gastric mill and pyloric rhythms. Two identified projection neurons [modulatory proctolin neuron (MPN) and modulatory commissural neuron 1 (MCN1)] that innervate the STG and modulate these rhythms contain GABA and the pentapeptide proctolin, but only MCN1 contains Cancer borealis tachykinin-related peptide (CabTRP Ia). Selective activation of each projection neuron elicits different rhythms from the STG. MPN elicits only a pyloric rhythm, whereas MCN1 elicits a distinct pyloric rhythm as well as a gastric mill rhythm. We tested the degree to which CabTRP Ia distinguishes the actions of MCN1 and MPN. To this end, we used the tachykinin receptor antagonist Spantide I to eliminate the actions of CabTRP Ia. With Spantide I present, MCN1 no longer elicited the gastric mill rhythm and the resulting pyloric rhythm was changed. Although this rhythm was more similar to the MPN-elicited pyloric rhythm, these rhythms remained different. Thus, CabTRP Ia partially confers the differences in rhythm generation resulting from MPN versus MCN1 activation. This result suggests that different projection neurons may use the same cotransmitters differently to elicit distinct pyloric rhythms. It also supports the hypothesis that different projection neurons use a combination of strategies, including using distinct cotransmitter complements, to elicit different outputs from the same neuronal network.

Distinct functions for cotransmitters mediating motor pattern selection.

Motor patterns are selected from multifunctional networks by selective activation of different projection neurons, many of which contain multiple transmitters. Little is known about how any individual projection neuron uses its cotransmitters to select a motor pattern. We address this issue by using the stomatogastric ganglion (STG) of the crab Cancer borealis, which contains a neuronal network that generates multiple versions of the pyloric and gastric mill motor patterns. The functional flexibility of this network results mainly from modulatory inputs it receives from projection neurons that originate in neighboring ganglia. We demonstrated previously that the STG motor pattern selected by activation of the modulatory proctolin neuron (MPN) results from direct MPN modulation of the pyloric rhythm and indirect MPN inhibition of the gastric mill rhythm. The latter action results from MPN inhibition of projection neurons that excite the gastric mill rhythm. These projection neurons are modulatory commissural neuron 1 (MCN1) and commissural projection neuron 2 (CPN2). MPN excitation of the pyloric rhythm is mimicked by bath application of proctolin, its peptide transmitter. Here, we show that MPN uses only its small molecule transmitter, GABA, to inhibit MCN1 and CPN2 within their ganglion of origin. We also demonstrate that MPN has no proctolin-mediated influence on MCN1 or CPN2, although exogenously applied proctolin directly excites these neurons. Thus, motor pattern selection occurs during MPN activation via proctolin actions on the STG network and GABA-mediated actions on projection neurons in the commissural ganglia, demonstrating a spatial and functional segregation of cotransmitter actions.

Coordination of fast and slow rhythmic neuronal circuits.

Interactions among rhythmically active neuronal circuits that oscillate at different frequencies are important for generating complex behaviors, yet little is known about the underlying cellular mechanisms. We addressed this issue in the crab stomatogastric ganglion (STG), which contains two distinct but interacting circuits. These circuits generate the gastric mill rhythm (cycle period, approximately 10 sec) and the pyloric rhythm (cycle period, approximately 1 sec). When the identified modulatory projection neuron named modulatory commissural neuron 1 (MCN1) is activated, the gastric mill motor pattern is generated by interactions among MCN1 and two STG neurons [the lateral gastric (LG) neuron and interneuron 1]. We show that, during MCN1 stimulation, an identified synapse from the pyloric circuit onto the gastric mill circuit is pivotal for determining the gastric mill cycle period and the gastric-pyloric rhythm coordination. To examine the role of this intercircuit synapse, we replaced it with a computational equivalent via the dynamic-clamp technique. This enabled us to manipulate better the timing and strength of this synapse. We found this synapse to be necessary for production of the normal gastric mill cycle period. The synapse acts, during each LG neuron interburst, to boost rhythmically the influence of the modulatory input from MCN1 to LG and thereby to hasten LG neuron burst onset. The two rhythms become coordinated because LG burst onset occurs with a constant latency after the onset of the triggering pyloric input. These results indicate that intercircuit synapses can enable an oscillatory circuit to control the speed of a slower oscillatory circuit, as well as provide a mechanism for intercircuit coordination.

Different proctolin neurons elicit distinct motor patterns from a multifunctional neuronal network.

Distinct motor patterns are selected from a multifunctional neuronal network by activation of different modulatory projection neurons. Subsets of these projection neurons can contain the same neuromodulator(s), yet little is known about the relative influence of such neurons on network activity. We have addressed this issue in the stomatogastric nervous system of the crab Cancer borealis. Within this system, there is a neuronal network in the stomatogastric ganglion (STG) that produces many versions of the pyloric and gastric mill rhythms. These different rhythms result from activation of different projection neurons that innervate the STG from neighboring ganglia and modulate STG network activity. Three pairs of these projection neurons contain the neuropeptide proctolin. These include the previously identified modulatory proctolin neuron and modulatory commissural neuron 1 (MCN1) and the newly identified modulatory commissural neuron 7 (MCN7). We document here that each of these neurons contains a unique complement of cotransmitters and that each of these neurons elicits a distinct version of the pyloric motor pattern. Moreover, only one of them (MCN1) also elicits a gastric mill rhythm. The MCN7-elicited pyloric rhythm includes a pivotal switch by one STG network neuron from playing a minor to a major role in motor pattern generation. Therefore, modulatory neurons that share a peptide transmitter can elicit distinct motor patterns from a common target network.

Frequency regulation of a slow rhythm by a fast periodic input.

Many nervous systems contain rhythmically active subnetworks that interact despite oscillating at widely different frequencies. The stomatogastric nervous system of the crab Cancer borealis produces a rapid pyloric rhythm and a considerably slower gastric mill rhythm. We construct and analyze a conductance-based compartmental model to explore the activation of the gastric mill rhythm by the modulatory commissural neuron 1 (MCN1). This model demonstrates that the period of the MCN1-activated gastric mill rhythm, which was thought to be determined entirely by the interaction of neurons in the gastric mill network, can be strongly influenced by inhibitory synaptic input from the pacemaker neuron of the fast pyloric rhythm, the anterior burster (AB) neuron. Surprisingly, the change of the gastric mill period produced by the pyloric input to the gastric mill system can be many times larger than the period of the pyloric rhythm itself. This model illustrates several mechanisms by which a fast oscillatory neuron may control the frequency of a much slower oscillatory network. These findings suggest that it is possible to modify the slow rhythm either by direct modulation or indirectly by modulating the faster rhythm.

Frequency control of a slow oscillatory network by a fast rhythmic input: pyloric to gastric mill interactions in the crab stomatogastric nervous system.

The stomatogastic nervous system of the crab, Cancer borealis, produces a slow gastric mill rhythm and a fast pyloric rhythm. When the gastric mill rhythm is not active, stimulation of the modulatory commissural ganglion neuron 1 (MCN1) activates a gastric mill rhythm in which the lateral gastric (LG) neuron fires in antiphase with interneuron 1 (Int1). We present theoretical and experimental data that indicate that the period of the MCN1 activated gastric mill rhythm depends on the strength and time course of the MCN1 evoked slow excitatory synaptic potential (EPSP) in the LG neuron, and on the strength of inhibition of Int 1 by the pacemaker of the pyloric network. This work demonstrates a new mechanism by which a slow network oscillator can be controlled by a much faster oscillatory neuron or network and suggests that modulation of the slow oscillator can occur by direct action on the neurons and synapses of the slow oscillator, or indirectly by actions on the fast oscillator and its synaptic connection with the slow oscillator.

Two novel tachykinin-related peptides from the nervous system of the crab Cancer borealis.

Immunocytochemical and biochemical studies have indicated the presence of many neuroactive substances in the stomatogastric nervous system (STNS) of the crab Cancer borealis. In electrophysiological studies, many of these substances modulate the motor output of neural networks contained within this system. Previous work in the STNS suggested the presence of neuropeptides related to the invertebrate tachykinin-related peptide (TRP) family. Here we isolate and characterize two novel peptides from the C. borealis nervous system that show strong amino acid sequence identity to the invertebrate TRPs. The central nervous systems of 160 crabs were extracted in an acidified solvent, after which four reversed-phase HPLC column systems were used to obtain pure peptides. A cockroach hindgut muscle contraction bioassay and a radioimmunoassay (RIA) employing an antiserum to locustatachykinin I (Lom TK I) were used to monitor all collected fractions. The amino acid sequences of the isolated peptides were determined by Edman degradation. Mass spectrometry and chemical synthesis confirmed the sequences to be APSGFLGMR-NH2 and SGFLGMR-NH2. APSGFLGMR-NH2 is approximately 20-fold more abundant in the crab central nervous system than is SGFLGMR-NH2. We have named these peptides Cancer borealis tachykinin-related peptide Ia and Ib (CabTRP Ia and Ib), respectively. Both peptides are myoactive in the cockroach hindgut muscle contraction bioassay, with CabTRP Ia being approximately 500 times more potent than CabTRP Ib. RIA performed on HPLC-separated C. borealis stomatogastric ganglion (STG) extract revealed that CabTRP Ia is the only detectable TRP-like moiety in this ganglion. Incubation of synthetic CabTRP Ia with the isolated STG excited the pyloric motor pattern. These effects were suppressed by the broad-spectrum tachykinin receptor antagonist Spantide I. Spantide I had no effect on the actions of the unrelated endogenous peptide proctolin in the STG. There was no consistent influence of CabTRP Ib on the pyloric rhythm. Given its amino acid sequence and minimal biological activity in the crab, CabTRP Ib may be a breakdown product of CabTRP Ia.

Motor pattern selection via inhibition of parallel pathways.

Motor pattern selection from a multifunctional neural network often results from direct synaptic and modulatory actions of different projection neurons onto neural network components. Less well documented is the presence and function of interactions among distinct projection neurons innervating the same network. In the stomatogastric nervous system of the crab Cancer borealis, several distinct projection neurons that influence the pyloric and gastric mill rhythms have been studied. These rhythms are generated by overlapping subsets of identified neurons in the stomatogastric ganglion (STG). One of these identified projection neurons is the modulatory proctolin neuron (MPN). We showed previously that MPN stimulation excites the pyloric rhythm by its excitatory actions on STG neurons. In contrast to its excitatory actions on the pyloric rhythm, we have now found that MPN inhibits the gastric mill rhythm. This inhibition does not occur within the STG, but instead results from MPN-mediated inhibition of two previously identified projection neurons within the commissural ganglia. These projection neurons innervate the STG and, via their actions on STG neurons, they elicit the gastric mill rhythm as well as modify the pyloric rhythm in a manner distinct from MPN. By inhibiting these projection neurons, MPN removes excitatory drive to gastric mill neurons and elicits an MPN-specific pyloric rhythm. Motor pattern selection by MPN therefore results from both a direct modulation of STG network activity and an inhibition of competing pathways.

Intercircuit control of motor pattern modulation by presynaptic inhibition.

Rhythmically active neural networks can control the modulatory input that they receive via their synaptic effects onto modulatory neurons. This synaptic control of network modulation can occur presynaptically, at the axon terminals of the modulatory neuron. For example, in the crab stomatogastric ganglion (STG), a gastric mill network neuron presynaptically inhibits transmitter release from a modulatory projection neuron called modulatory commissural neuron 1. We showed previously that the gastric mill rhythm-timed presynaptic inhibition of the STG terminals of MCN1 is pivotal for enabling MCN1 to activate this rhythm. We also showed that MCN1 excites the pyloric rhythm within the STG. Here we show that, because MCN1 stimulation conjointly excites the gastric mill and pyloric rhythms, the gastric mill rhythm-timed presynaptic inhibition of MCN1 causes a rhythmic interruption in the MCN1-mediated excitation of the pyloric rhythm. Consequently, during each protraction phase of the gastric mill rhythm, presynaptic inhibition suppresses MCN1 excitation of the pyloric rhythm, thereby weakening the pyloric rhythm. During the retraction phase, presynaptic inhibition is absent and MCN1 elicits a faster, stronger, and modified pyloric rhythm. Thus, in addition to its role in enabling a neural circuit to regulate the modulatory transmission that it receives, presynaptic inhibition is also used effectively to rhythmically control the activity level of a distinct, but behaviorally related, neural circuit.

Pyloric motor pattern modification by a newly identified projection neuron in the crab stomatogastric nervous system.

1. We have used multiple, simultaneous intra- and extracellular recordings as well as Lucifer yellow dye-fills to identify modulatory commissural neuron 5 (MCN5) and characterize its effects in the stomatogastric nervous system (STNS) of the crab, Cancer borealis. MCN5 has a soma and neuropilar arborization in the commissural ganglion (CoG; Figs. 1 and 2), and it projects through the inferior esophageal nerve (ion) and stomatogastric nerve (stn) to the stomatogastric ganglion (STG; Figs. 1-3). 2. Within the CoGs, MCN5 receives esophageal rhythm-timed excitation and pyloric rhythm-timed inhibition (Fig. 4). Additionally, during the lateral teeth protractor phase of the gastric mill rhythm, the pyloric-timed inhibition of MCN5 is reduced or eliminated. 3. Intracellular stimulation of MCN5 excites the pyloric pacemaker ensemble, including the anterior burster (AB), pyloric dilator (PD), and lateral posterior gastric (LPG) neurons. This produces a faster pyloric rhythm. MCN5 stimulation also inhibits all nonpacemaker pyloric neurons, reducing or eliminating their activity (Figs. 5 and 6A; Tables 1 and 2). After MCN5 stimulation, bursting is enhanced for several cycles in some pyloric neurons when compared with their prestimulus activity (Figs. 5 and 6A; Tables 1 and 2). 4. MCN5 evokes distinct responses from each pyloric pacemaker neuron (Figs. 6-8). The AB and LPG neurons respond with increased activity. The AB response includes the presence of large amplitude excitatory postsynaptic potentials (EPSPs) that contribute to a depolarization of the trough of its rhythmic oscillations (Fig. 6). LPG responds by exhibiting increased activity that prolongs the duration of its burst beyond that of AB and PD (Fig. 7). In contrast, MCN5 stimulation initially produces decreased PD neuron activity, followed by a slight enhancement of each PD burst (Figs. 7 and 8). PD activity is further enhanced after MCN5 stimulation (Figs. 7 and 8). 5. MCN5-elicited action potentials evoke discrete, constant latency inhibitory postsynaptic potentials (IPSPs) in all nonpacemaker pyloric neurons, including the inferior cardiac (IC), lateral pyloric (LP), pyloric (PY), and ventricular dilator (VD) neurons (Fig. 9). MCN5 activity also inhibits these neurons indirectly, via its excitation of the pacemaker neurons. The pyloric pacemaker neurons synaptically inhibit all four nonpacemaker neurons. 6. The increased activity in the VD neuron, after MCN5 stimulation, is not mimicked by either direct hyperpolarization or by synaptically inhibiting VD via another pathway (Fig. 10). The poststimulation increase in IC neuron activity is stronger than that after hyperpolarizing current injection but is comparable with that resulting from stimulation of another inhibitory pathway (Fig. 10). The enhanced PY neuron activity is comparable with that resulting from either direct current injection or synaptic inhibition from another pathway (Fig. 10). 7. MCN5 activity increases the pyloric cycle frequency of both slow (< 1 Hz) and fast (1-2 Hz) rhythms (Fig. 11), and it significantly alters the phase relationships that define this motor pattern (Fig. 12). These phase relationships change again after MCN5 stimulation (Fig. 12). 8. MCN5 acts in concert with the pyloric pacemaker ensemble to elicit a pyloric rhythm that exhibits enhanced pacemaker neuron activity and reduced activity in all nonpacemaker neurons. Additionally, despite their electrical coupling, the three types of pacemaker neurons exhibit distinct responses to MCN5 stimulation. This partially uncouples their normally coactive bursts. The resulting motor pattern is distinct from all previously characterized pyloric rhythms.

A switch between two modes of synaptic transmission mediated by presynaptic inhibition.

Presynaptic inhibition reduces chemical synaptic transmission in the central nervous system between pairs of neurons, but its role(s) in shaping the multisynaptic interactions underlying neural network activity are not well studied. We therefore used the crustacean stomatogastric nervous system to study how presynaptic inhibition of the identified projection neuron, modulatory commissural neuron 1 (MCN1), influences the MCN1 synaptic effects on the gastric mill neural network. Tonic MCN1 discharge excites gastric mill network neurons and activates the gastric mill rhythm. One network neuron, the lateral gastric (LG) neuron, presynaptically inhibits MCN1 and is electrically coupled to its terminals. We show here that this presynaptic inhibition selectively reduces or eliminates transmitter-mediated excitation from MCN1 without reducing its electrically mediated excitatory effects, thereby switching the network neurons excited by MCN1. By switching the type of synaptic output from MCN1 and, hence, the activated network neurons, this presynaptic inhibition is pivotal to motor pattern generation.

Distribution and effects of tachykinin-like peptides in the stomatogastric nervous system of the crab, Cancer borealis.

The rhythmically active pyloric and gastric mill motor patterns in the stomatogastric ganglion of the crab, Cancer borealis, are influenced by modulatory projection neurons whose somata are located primarily in the other ganglia of the stomatogastric nervous system. One of these projection neurons exhibits substance P-like immunolabeling. However, bath application of substance P does not influence these motor patterns. To determine whether a different peptide is responsible for the substance P-like immunolabeling, we studied the presence and physiological effects of the locustatachykinins and the leucokinins, two families of tachykinin-like peptides originally identified in insect nervous systems. Locustatachykinin-like immunolabeling has the same distribution in the stomatogastric nervous system as substance P-like immunolabeling and colocalizes with it in the majority of immunopositive structures. Preincubation of locustatachykinin antibody with substance P, and preincubation of substance P antibody with locustatachykinin, blocks subsequent immunolabeling in the stomatogastric nervous system. In contrast, we found no leucokinin-like immunolabeling in this system. Bath application to the stomatogastric ganglion of individual locustatachykinins or leucokinins excited the pyloric rhythm in a state-dependent manner. Each peptide family had distinct effects on the pyloric rhythm. Thus, both of these tachykinin-like peptide families are likely related to native neuropeptides that influence the pyloric rhythm. Furthermore, a member of the locustatachykinin family is likely to be the source of the previously identified substance P-like immunoreactivity in the stomatogastric nervous system.

Presynaptic control of neurones in pattern-generating networks.

Recent studies have revealed presynaptic influences on neurones that participate in rhythmic motor patterns. Although there is still little direct information about the effects of these inputs at presynaptic terminals, their functional consequences are being unraveled. These presynaptic influences gate sensory input to pattern-generating networks and locally alter the synaptic strength and/or the activity pattern of network neurones.

Functional consequences of compartmentalization of synaptic input.

Intra-axonal recordings of stomatogastric nerve axon 1 (SNAX1) indicate that there are synaptic inputs onto the SNAX1 terminals in the stomatogastric ganglion (STG) of the crab Cancer borealis (Nusbaum et al., 1992b). To determine whether this synaptic input only influenced SNAX1 activity within the STG, we identified the SNAX1 soma in the commissural ganglion (CoG). We found that this neuron has a neuropilar arborization in the CoG and also receives synaptic inputs in this ganglion. Based on its soma location, we have renamed this neuron modulatory commissural neuron 1 (MCN1). While intracellular stimulation of MCN1soma and MCN1SNAX has the same excitatory effects on the STG motor patterns, MCN1 receives distinct synaptic inputs in the STG and CoG. Moreover, the synaptic input that MCN1 receives within the STG compartmentalizes its activity. Specifically, the lateral gastric (LG) neuron synaptically inhibits MCN1SNAX-initiated activity within the STG (Nusbaum et al., 1992b), and while LG did not inhibit MCN1soma-initiated activity in the CoG, it did inhibit these MCN1 impulses when they arrived in the STG. As a result, during MCN1soma-elicited gastric mill rhythms, MCN1soma is continually active in the CoG but its effects are rhythmically inhibited in the STG by LG neuron impulse bursts. One functional consequence of this local control of MCN1 within the STG is that the LG neuron thereby controls the timing of the impulse bursts in other gastric mill neurons. Thus, local synaptic input can functionally compartmentalize the activity of a neuron with arbors in distinct regions of the nervous system.

Neuropeptide degradation produces functional inactivation in the crustacean nervous system.

The pentapeptide proctolin (Proct.; Arg-Tyr-Leu-Pro-Thr) is a modulatory transmitter found throughout the crustacean nervous system. No information is available in this system, however, as to how the actions of this peptide are terminated. To study this issue in the crab Cancer borealis, we incubated exogenous proctolin (10(-5) M) with either the thoracic ganglion (TG) or with conditioned saline (CS) that had been preincubated with the TG. We removed aliquots at standard time points for analysis by reverse-phase high-performance liquid chromatography (HPLC). We found that over time the proctolin peak became progressively smaller, while three novel peaks appeared and increased in size. Comigration experiments using HPLC indicated that the major novel peak was Proct. (Tyr-Leu-Pro-Thr), while one of the two minor peaks was Proct. (Leu-Pro-Thr). The other minor peak appeared to be Proct. (Arg-Tyr), based on similar HPLC retention time to synthetic Proct. The reduction in the proctolin peak and the increase in the Proct. peak was prevented by co-incubation of proctolin with any one of several aminopeptidase inhibitors (10(-4) M). Proct. and Proct. appeared to result from a diaminopeptidase-mediated cleavage of proctolin. We tested whether N-terminal cleavage functionally inactivated proctolin by coapplying proctolin (10(-8) M) and individual aminopeptidase inhibitors (10(-5) M) to the isolated stomatogastric ganglion (STG). We found that these inhibitors significantly enhanced the proctolin excitation of the pyloric rhythm. Furthermore, application of synthetic Proct. to the STG had no effect unless high concentrations (> 10(-6) M) were used, and neither Proct. nor Proct. (10(-4) M) influenced the pyloric rhythm. Our results indicate that proctolin is enzymatically degraded and thereby biologically inactivated in the crab nervous system, primarily by extracellularly located aminopeptidase activity.

Recruitment of a projection neuron determines gastric mill motor pattern selection in the stomatogastric nervous system of the crab, Cancer borealis.

1. In the isolated stomatogastric nervous system of the crab Cancer borealis (Fig. 1), the muscarinic agonist oxotremorine elicits several distinct gastric mill motor patterns from neurons in the stomatogastric ganglion (STG; Fig. 2). Selection of a particular gastric mill rhythm is determined by activation of distinct projection neurons that influence gastric mill neurons within the STG. In this paper we identify one such neuron, called commissural projection neuron 2 (CPN2), whose rhythmic activity is integral in producing one form of the gastric mill rhythm. 2. There is a CPN2 soma and neuropilar arborization in each commissural ganglion (CoG). The CPN2 axon projects through the superior esophageal nerve (son) and the stomatogastric nerve (stn) to influence neurons in the STG (Figs. 3 and 4A). 3. CPN2 activity influences most of the gastric mill neurons in the STG. Specifically, CPN2 excites gastric mill neurons GM and LG (gastric mill and lateral gastric, respectively) and inhibits the dorsal gastric (DG), anterior median (AM), medial gastric (MG), and inferior cardiac (IC) neurons (Figs. 5 and 6). CPN2 also indirectly inhibits gastric mill neurons Int1 and VD (interneuron 1 and ventricular dilator neuron, respectively) through its activation of LG. The CPN2 excitatory effects are mediated at least partly via discrete excitatory postsynaptic potentials (EPSPs; Fig. 4B), whereas its inhibitory effects are produced via smooth hyperpolarizations. 4. Within the CoG, CPN2 receives excitatory synaptic input from the anterior gastric receptor neuron (AGR), a gastric mill proprioceptive sensory neuron (Fig. 7) and inhibitory synaptic input from the gastric mill interneuron, Int1 (Fig. 8). 5. During one form of the gastric mill rhythm, CPN2 fires rhythmically in time with the gastric mill motor pattern, whereas it is silent or fires weakly during other gastric mill rhythms (Fig. 9). 6. When CPN2 rhythmic activity is suppressed during a CPN2-influenced gastric mill rhythm, the gastric mill rhythm continues, but the pattern is altered (Fig. 10). Moreover, transiently stimulating CPN2 during any ongoing gastric mill motor pattern can reset the timing of that rhythm (Fig. 11). 7. Tonic activity in CPN2 is insufficient to elicit a gastric mill rhythm (Fig. 12). Phasic activity in CPN2 can elicit a gastric mill rhythm only in preparations in which gastric mill neurons are already in an excited state (Figs. 12 and 13). 8. CPN2 recruitment plays a pivotal role in determining the final form of the gastric mill rhythm.(ABSTRACT TRUNCATED AT 400 WORDS)