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.     

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.     

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.     

Motor pattern selection by nitric oxide in the stomatogastric nervous system of the crab

The gas nitric oxide (NO) serves a diversity of functions in the nervous system and plays an important role in the modulation of oscillatory networks. We investigated the actions of intrinsically produced NO on the rhythmically active gastric mill circuit within the stomatogastric ganglion (STG) of the crab, Cancer pagurus. Bath application of different NO blockers exclusively to the STG terminated spontaneously active gastric mill rhythms. Furthermore, a reduction in the activity levels of projection neurons that sustain the gastric mill rhythm was observed, suggesting that NO blockade influences feedback mechanisms that affect projection neuron activity. When STG feedback to these projection neurons was intact, their activity decreased strongly with NO blockers present exclusively in the STG. When either neuronal feedback was eliminated or projection neurons were tonically activated, NO blockade did not terminate the gastric mill rhythm, indicating an indirect ascending control of the projection neurons. Together, our results show that ascending feedback from a motor network is important in shaping network activity and that this feedback is state-dependent and can be modulated to alter the output of the motor network.     

Actions of a histaminergic/peptidergic projection neuron on rhythmic motor patterns in the stomatogastric nervous system of the crab Cancer borealis

Histamine is a neurotransmitter with actions throughout the nervous system of vertebrates and invertebrates. Nevertheless, the actions of only a few identified histamine-containing neurons have been characterized. Here, we present the actions of a histaminergic projection neuron on the rhythmically active pyloric and gastric mill circuits within the stomatogastric ganglion (STG) of the crab Cancer borealis. An antiserum generated against histamine labeled profiles throughout the C. borealis stomatogastric nervous system. Labeling occurred in several somata and neuropil within the paired commissural ganglia as well as in neuropil within the STG and at the junction of the superior oesophageal and stomatogastric nerves. The source of all histamine-like immunolabeling in the STG neuropil was one pair of neuronal somata, the previously identified inferior ventricular (IV) neurons, located in the supraoesophageal ganglion. These neurons also exhibited FLRFamide-like immunoreactivity. Activation of the IV neurons in the crab inhibited some pyloric and gastric mill neurons and, with inputs from the commissural ganglia eliminated, terminated both rhythms. Focal application of histamine had comparable effects. The actions of both applied histamine and IV neuron stimulation were blocked, reversibly, by the histamine type-2 receptor antagonist cimetidine. With the commissural ganglia connected to the STG, IV neuron stimulation elicited a longer-latency activation of commissural projection neurons which in turn modified the pyloric rhythm and activated the gastric mill rhythm. These results support the hypothesis that the histaminergic/peptidergic IV neurons are projection neurons with direct and indirect actions on the STG circuits of the crab C. borealis.     

Regulating peptidergic modulation of rhythmically active neural circuits

The ability of neuropeptides to modulate neural circuit activity is well established, but little is known regarding how the actions of neurally-released peptides are regulated. This issue is being studied in the isolated stomatogastric nervous system (STNS) of decapod crustaceans. The STNS is a small neural system that contains the rhythmically active gastric mill (chewing) and pyloric (filtering of chewed food) motor circuits within the stomatogastric ganglion (STG). These circuits are influenced by a set of modulatory projection neurons in the neighboring commissural and oesophageal ganglia. This system includes three different projection neurons that contain the peptide transmitter proctolin among an overlapping complement of cotransmitters. Despite their shared proctolinergic phenotype, when these projection neurons are activated individually each of them has distinct actions on the gastric mill and pyloric circuits. These distinct actions result only partly from the presence of different cotransmitters in these projection neurons. Also contributing to these distinct actions are differences in the pattern of transmitter release as well as a differential, peptidase-mediated sculpting of the actions of the proctolin released from each projection neuron. There is also a convergence of peptide cotransmitter actions, at the level of the target ion channel, which might limit the effectiveness of each individual cotransmitter. One lesson already learned from this small neural system is that there is a diverse collection of regulatory mechanisms for controlling the actions of neurally-released peptides on rhythmically active neural circuits.     

Neuropeptide Modulation Increases Dendritic Electrical Spread to Restore Neuronal Activity Disrupted by Temperature

Peptide neuromodulation has been implicated to shield neuronal activity from acute temperature changes that can otherwise lead to loss of motor control or failure of vital behaviors. However, the cellular actions neuropeptides elicit to support temperature-robust activity remain unknown. Here, we find that peptide neuromodulation restores rhythmic bursting in temperature-compromised central pattern generator (CPG) neurons by counteracting membrane shunt and increasing dendritic electrical spread. We show that acutely rising temperatures reduced spike generation and interrupted ongoing rhythmic motor activity in the crustacean gastric mill CPG. Neuronal release and extrinsic application of Cancer borealis tachykinin-related peptide Ia (CabTRP Ia), a substance-P-related peptide, restored rhythmic activity. Warming led to a significant decrease in membrane resistance and a shunting of the dendritic signals in the main gastric mill CPG neuron. Using a combination of fluorescent calcium imaging and electrophysiology, we observed that postsynaptic potentials and antidromic action potentials propagated less far within the dendritic neuropil as the system warmed. In the presence of CabTRP Ia, membrane shunt decreased and both postsynaptic potentials and antidromic action potentials propagated farther. At elevated temperatures, CabTRP Ia restored dendritic electrical spread or extended it beyond that at cold temperatures. Selective introduction of the CabTRP Ia conductance using a dynamic clamp demonstrated that the CabTRP Ia voltage-dependent conductance was sufficient to restore rhythmic bursting. Our findings demonstrate that a substance-P-related neuropeptide can boost dendritic electrical spread to maintain neuronal activity when perturbed and reveals key neurophysiological components of neuropeptide actions that support pattern generation in temperature-compromised conditions.SIGNIFICANCE STATEMENT Changes in body temperature can have detrimental consequences for the well-being of an organism. Temperature-dependent changes in neuronal activity can be especially dangerous if they affect vital behaviors. Understanding how temperature changes disrupt neuronal activity and identifying how to ameliorate such effects is critically important. Our study of a crustacean circuit shows that warming disrupts rhythmic neuronal activity by increasing membrane shunt and reducing dendritic electrical spread in a key circuit neuron. Through the ionic conductance activated by it, substance-P-related peptide modulation restored electrical spread and counteracted the detrimental temperature effects on rhythmic activity. Because neuropeptides are commonly implicated in sustaining neuronal activity during perturbation, our results provide a promising mechanism to support temperature-robust activity.     

Presynaptic control of modulatory fibers by their neural network targets.

Numerous modulatory fibers control the output of the pyloric and gastric mill neural networks in the crustacean stomatogastric ganglion (STG). We now describe the first results of intracellular recordings from the axon of one of these input neurons, stomatogastric nerve axon 1 (SNAX 1), close to where it enters the STG. SNAX 1 excites both the pyloric and gastric mill rhythms and is identified on the basis of its synaptic interactions with identified STG neurons. SNAX 1 receives synaptic input from several sources within the STG. As a result of these synaptic inputs, SNAX 1 fires bursts of action potentials that are time-locked to both the pyloric and gastric mill rhythms. The synaptic connections made onto the SNAX axon terminals are likely to play important roles in shaping the impulse activity patterns in these modulatory inputs. Thus, the fibers that modulate the pattern-generating networks in the STG are themselves influenced by elements in these networks, and modulation is a dynamic interaction between input fibers and STG neurons.     

A modulatory proctolin-containing neuron (MPN). I. Identification and characterization

The pentapeptide proctolin has been localized previously to the crustacean stomatogastric nervous system and shown to modulate the rhythmic activity of the pyloric network in the stomatogastric ganglion (STG) (Marder et al., 1986; Hooper and Marder, 1987). We have now identified a pair of modulatory proctolin-containing neurons (MPNs) that cause proctolin-like modulation of the pyloric rhythm. Individual MPNs were identified by combining intracellular Lucifer yellow dye injection with rhodamine-visualized proctolin immunolabeling. Both MPNs are located in the esophageal nerve and send processes to the STG. Current injection into one MPN influences the second MPN, suggesting that they are electrically coupled. The 2 MPNs have similar effects on the pyloric rhythm of the STG. Intracellular stimulation of a single MPN was sufficient to enhance already active pyloric rhythms and initiated the pyloric rhythm in quiescent preparations.     

Network oscillations generated by balancing graded asymmetric reciprocal inhibition in passive neurons.

We describe a novel mechanism by which network oscillations can arise from reciprocal inhibitory connections between two entirely passive neurons. The model was inspired by the activation of the gastric mill rhythm in the crab stomatogastric ganglion by the modulatory commissural ganglion neuron 1 (MCN1), but it is studied here in general terms. One model neuron has a linear current-voltage (I-V) curve with a low (L) resting potential, and the second model neuron has a linear current-voltage curve with a high (H) resting potential. The inhibitory connections between them are graded. There is an extrinsic modulatory excitatory input to the L neuron, and the L neuron presynaptically inhibits the modulatory neuron. Activation of the extrinsic modulatory neuron elicits stable network oscillations in which the L and H neurons are active in alternation. The oscillations arise because the graded reciprocal synapses create the equivalent of a negative-slope conductance region in the I-V curves for the cells. Geometrical methods are used to analyze the properties of and the mechanism underlying these network oscillations.     

Neuronal Switching between Single- and Dual-Network Activity via Modulation of Intrinsic Membrane Properties

Oscillatory networks underlie rhythmic behaviors (e.g., walking, chewing) and complex behaviors (e.g., memory formation, decision-making). Flexibility of oscillatory networks includes neurons switching between single- and dual-network participation, even generating oscillations at two distinct frequencies. Modulation of synaptic strength can underlie this neuronal switching. Here we ask whether switching into dual-frequency oscillations can also result from modulation of intrinsic neuronal properties. The isolated stomatogastric nervous system of male Cancer borealis crabs contains two well-characterized rhythmic feeding-related networks (pyloric, ∼1 Hz; gastric mill, ∼0.1 Hz). The identified modulatory projection neuron MCN5 causes the pyloric-only lateral posterior gastric (LPG) neuron to switch to dual pyloric/gastric mill bursting. Bath applying the MCN5 neuropeptide transmitter Gly¹-SIFamide only partly mimics the LPG switch to dual activity because of continued LP neuron inhibition of LPG. Here, we find that MCN5 uses a cotransmitter, glutamate, to inhibit LP, unlike Gly¹-SIFamide excitation of LP. Thus, we modeled the MCN5-elicited LPG switching with Gly¹-SIFamide application and LP photoinactivation. Using hyperpolarization of pyloric pacemaker neurons and gastric mill network neurons, we found that LPG pyloric-timed oscillations require rhythmic electrical synaptic input. However, LPG gastric mill-timed oscillations do not require any pyloric/gastric mill synaptic input and are voltage-dependent. Thus, we identify modulation of intrinsic properties as an additional mechanism for switching a neuron into dual-frequency activity. Instead of synaptic modulation switching a neuron into a second network as a passive follower, modulation of intrinsic properties could enable a switching neuron to become an active contributor to rhythm generation in the second network.SIGNIFICANCE STATEMENT Neuromodulation of oscillatory networks can enable network neurons to switch from single- to dual-network participation, even when two networks oscillate at distinct frequencies. We used small, well-characterized networks to determine whether modulation of synaptic strength, an identified mechanism for switching, is necessary for dual-network recruitment. We demonstrate that rhythmic electrical synaptic input is required for continued linkage with a “home” network, whereas modulation of intrinsic properties enables a neuron to generate oscillations at a second frequency. Neuromodulator-induced switches in neuronal participation between networks occur in motor, cognitive, and sensory networks. Our study highlights the importance of considering intrinsic properties as a pivotal target for enabling parallel participation of a neuron in two oscillatory networks.     

Multiple peptides converge to activate the same voltage-dependent current in a central pattern-generating circuit.

The stomatogastric ganglion of the crab, Cancer borealis, is modulated by >20 different substances, including numerous neuropeptides. One of these peptides, proctolin, activates an inward current that shows strong outward rectification (Golowasch and Marder, 1992). Decreasing the extracellular Ca(2+) concentration linearizes the current-voltage curve of the proctolin-induced current. We used voltage clamp to study the currents evoked by proctolin and five additional modulators [C. borealis tachykinin-related peptide Ia (CabTRP Ia), crustacean cardioactive peptide, red pigment-concentrating hormone, TNRNFLRFamide, and the muscarinic agonist pilocarpine] in stomatogastric ganglion neurons, both in the intact ganglion and in dissociated cell culture. Subtraction currents yielded proctolin-like current-voltage relationships for all six substances, and the current-voltage curves of all six substances showed linearization in low external Ca(2+). The lateral pyloric neuron responded to all six modulators, but the ventricular dilator neuron only responded to a subset of them. Bath application of saturating concentrations of proctolin occluded the response to CabTRP and vice versa. N-(6-Aminohexyl)-5-chloro-1-napthalensulfonamide, a calmodulin inhibitor, increased the amplitude and altered the voltage dependence of the responses elicited by CabTRP and proctolin. Together, these data indicate that all six substances converge onto the same voltage-dependent current, although they activate different receptors. Therefore, differential network responses evoked by these substances may primarily depend on the receptor distribution on network neurons.     

Reconfiguration of multiple motor networks by short- and long-term actions of an identified modulatory neuron

The pyloric and gastric motor pattern-generating networks in the stomatogastric ganglion of the lobster Homarus gammarus are reconfigured into a new functional circuit by burst discharge in an identified pair of modulatory projection interneurons, originally named the pyloric suppressor (PS) neurons because of their inhibitory effects on pyloric network activity. Here we elucidate the actions of the PS neurons on individual members of the neighbouring gastric circuit, as well as describing their ability to alter synaptic coupling between the two networks. PS neuron firing has two distinct effects on gastric network activity: an initial short-lasting action mediated by transient inhibition of most gastric motoneurons, followed by a long-lasting circuit activation associated with a prolonged PS-evoked depolarization of the medial gastric (MG) motoneuron and the single network interneuron, Int1. These long-lasting effects are voltage-dependent, and experiments with hyperpolarizing current injection and photoablation suggest that excitation of both the MG neuron and Int1 is critical for PS-elicited gastric network rhythmicity. In parallel, PS neuron discharge persistently (lasting several minutes) enhances the strength of an inhibitory synaptic influence of the MG neuron on the pyloric dilator (PD)-anterior burster (AB) pacemaker neurons, thereby facilitating operational fusion of the two networks. Therefore, a single modulatory neuron may influence disparate populations of neurons via a range of very different and highly target-specific mechanisms: conventional transient synaptic drive and up- or down-modulation of membrane properties and synaptic efficacy. Moreover, distinctly different time courses of these actions allow different circuit configurations to be specified sequentially by a given modulatory input.     

Discovery and functional study of a novel crustacean tachykinin neuropeptide

Tachykinin-related peptide (TRP) refers to a large and structurally diverse family of neuropeptides found in vertebrate and invertebrate nervous systems. These peptides have various important physiological functions, from regulating stress in mammals to exciting the pyloric (food filtering) rhythm in the stomatogastric nervous system (STNS) of decapod crustaceans. Here, a novel TRP, which we named CalsTRP (Callinectes sapidus TRP), YPSGFLGMRamide (m/z 1026.52), was identified and de novo sequenced using a multifaceted mass spectrometry-based platform in both the central nervous system (CNS) and STNS of C. sapidus. We also found, using isotopic formaldehyde labeling, that CalsTRP in the C. sapidus brain and commissural ganglion (CoG) was up-regulated after food-intake, suggesting that TRPs in the CNS and STNS are involved in regulating feeding in Callinectes. Using imaging mass spectrometry, we determined that the previously identified CabTRP Ia (APSGFLGMRamide) and CalsTRP were co-localized in the C. sapidus brain. Lastly, our electrophysiological studies show that bath-applied CalsTRP and CabTRP Ia each activates the pyloric and gastric mill rhythms in C. sapidus, as shown previously for pyloric rhythm activation by CabTRP Ia in the crab Cancer borealis. In summary, the newly identified CalsTRP joins CabTRP Ia as a TRP family member in the decapod crustacean nervous system, whose actions include regulating feeding behavior.     

Modulation of circuit feedback specifies motor circuit output

Bidirectional communication (i.e., feedforward and feedback pathways) between functional levels is common in neural systems, but in most systems little is known regarding the function and modifiability of the feedback pathway. We are exploring this issue in the crab (Cancer borealis) stomatogastric nervous system by examining bidirectional communication between projection neurons and their target central pattern generator (CPG) circuit neurons. Specifically, we addressed the question of whether the peptidergic post-oesophageal commissure (POC) neurons trigger a specific gastric mill (chewing) motor pattern in the stomatogastric ganglion solely by activating projection neurons, or by additionally altering the strength of CPG feedback to these projection neurons. The POC-triggered gastric mill rhythm is shaped by feedback inhibition onto projection neurons from a CPG neuron. Here, we establish that POC stimulation triggers a long-lasting enhancement of feedback-mediated IPSC/Ps in the projection neurons, which persists for the same duration as POC-gastric mill rhythms. This strengthened CPG feedback appears to result from presynaptic modulation, because it also occurs in other projection neurons whose activity does not change after POC stimulation. To determine the function of this strengthened feedback synapse, we compared the influence of dynamic-clamp-injected feedback IPSPs of pre- and post-POC amplitude into a pivotal projection neuron after POC stimulation. Only the post-POC amplitude IPSPs elicited the POC-triggered activity pattern in this projection neuron and enabled full expression of the POC-gastric mill rhythm. Thus, the strength of CPG feedback to projection neurons is modifiable and can be instrumental to motor pattern selection.     

A modulatory proctolin-containing neuron (MPN). II. State-dependent modulation of rhythmic motor activity

The effects of stimulating the modulatory proctolin-containing neurons (MPNs) on the pyloric rhythm of the stomatogastric ganglion of the crab, Cancer borealis, were compared with those produced by exogenously applied proctolin. The effects of both MPN stimulation and proctolin applications depend on the preceding physiological state of the preparation. Both treatments increase the pyloric cycle frequency dramatically in preparations that are slowly cycling, but they have little or no effect on pyloric cycle frequency in preparations that are already rapidly cycling. MPN and proctolin produce maximal pyloric cycle frequencies of about 1.2 Hz, although much faster pyloric frequencies are possible. MPN stimulation and proctolin applications affect the number of impulses fired in each burst by pyloric network neurons. MPN's excitatory actions are longer lasting when a preparation is active than when it is quiescent before stimulation. These data suggest that many of MPN's physiological actions result from its release of proctolin. Small unitary postsynaptic potentials evoked by MPN stimulation in the lateral pyloric neuron may indicate the presence of a second neurotransmitter in MPN.     

Dynamic restructuring of a rhythmic motor program by a single mechanoreceptor neuron in lobster.

We have explored the synaptic and cellular mechanisms by which a single primary mechanosensory neuron, the anterior gastric receptor (AGR), reconfigures motor output of the gastric mill central pattern generator (CPG) in the stomatogastric nervous system (STNS) of the lobster Homarus gammarus. AGR is activated in vivo by contraction of the medial tooth protractor muscle gm1 and accesses the gastric CPG via excitation of two in-parallel interneurons, the excitatory commissural gastric (CG) and the inhibitory gastric inhibitor (GI). In the spontaneously active STNS in vitro, weak firing of AGR in time with gastric mill motoneurons (GM) reinforces an ongoing type I gastric mill rhythm in which all gastric teeth power-stroke motoneurons are synchronously active. With strong AGR firing, these phase relationships switch abruptly to a type II pattern in which lateral and medial teeth power-stroke motoneurons fire in antiphase. Our results suggest that these bimodal actions on the gastric mill rhythm depend on the balance of firing of the CG and GI interneurons and that selection of the pathway resides in their different postsynaptic sensitivities to AGR. Whereas high intrinsic firing rates of the CG neuron ensure that the excitatory pathway predominates during low levels of sensory input, strong synaptic facilitation in the GI neuron favors the inhibitory pathway during high levels of receptor activity. Feedback from a single mechanosensory neuron is thus able, in an activity-dependent manner, to specify different motor programs from a single central pattern-generating network.     

Multiple axonal spike initiation zones in a motor neuron: serotonin activation.

The lateral gastric (LG) motor neuron of the stomatogastric nervous system of the crab Cancer borealis has a large soma in the stomatogastric ganglion (STG). The LG motor neuron makes inhibitory synaptic connections within the neuropil of the STG, and also projects to the periphery, where it innervates a series of muscles that control the movements of the lateral teeth of the gastric mill. The LG motor neuron has a spike initiation zone close to its neuropilar integrative regions, from which spikes propagate orthodromically to the muscles. Additionally, under certain conditions, the LG neuron can initiate spikes at peripheral axonal sites that can be 0.5-2.0 cm from the STG. Peripherally initiated spikes propagate antidromically into the STG and also propagate to the muscle. The peripheral spike initiation zones are often active in combined preparations in which the muscles are left attached. When the muscles are removed, depolarization of the LG soma together with 5-HT applied to the motor nerve also evokes peripheral spike initiation. At a given 5-HT concentration, the duration of the trains of antidromic spikes can be controlled by current injection into the soma, suggesting the presence of a slow voltage-dependent conductance in the LG axon. The antidromic spikes contribute to lengthening of the duration of contraction in some of the muscles innervated by the LG, but do not evoke IPSPs onto LG follower neurons. Thus, the LG neuron can send different signals to its peripheral and central targets.     

Ontogenetic alteration in peptidergic expression within a stable neuronal population in lobster stomatogastric nervous system

In the adult lobster, Homarus gammarus, the stomatogastric ganglion (STG) contains two well-defined motor pattern generating networks that receive numerous modulatory peptidergic inputs from anterior ganglia. We are studying the appearance of extrinsic peptidergic inputs to these networks during ontogenesis. Neuron counts indicate that as early as 20% of development (E20) the STG neuronal population is quantitatively established. By using immunocytochemical detection of 5-bromo-2′-deoxyuridine incorporation, we found no immunopositive cells in the STG by E70. We concluded that the STG neuronal population remains quantitatively stable from mid-embryonic life until adulthood. We then investigated the ontogeny of FLRFamide- and proctolin-like peptides in the stomatogastric nervous system, from their first appearance until adulthood by using whole mount immunocytochemistry. Numerous FLRFamide-like-immunoreactive STG neuropilar ramifications were observable as early as E45 and remain thereafter. From E50 to the first larval stage, one to three STG somata stained, while somatic staining was not observed in larval stage II and subsequent stages. From E50 and thereafter, the STG neuropilar area was immunopositive for proctolin. One to two proctolinergic somata were detected in the STG of the three larval stages but were not seen in embryos, the post-larval stage or in adults. Thus, peptidergic inputs to the STG are present from mid-embryonic life. Moreover, whereas in the adult, STG neurons only contain glutamate or acetylcholine, some neurons transiently express peptidergic phenotypes during development. Although this system expresses an ontogenetic peptidergic plasticity, the STG neurons produce a single stable embryonic-larval motor output (Casasnovas and Meyrand [1995] J. Neurosci. 15:5703–5718). J. Comp. Neurol. 399:289–305, 1998. © 1998 Wiley-Liss, Inc.     

Cholecystokinin-like peptide is a modulator of a crustacean central pattern generator

The presence, release, and physiological effects of a cholecystokinin(CCK)-like peptide within the stomatogastric ganglion (STG) of the lobster, Panulirus interruptus, are described. Indirect immunofluorescence with 2 antisera raised against CCK8 was used to determine the distribution of CCK-like immunoreactivity (CCKLI) in the stomatogastric nervous system. CCKLI was demonstrated in the input nerve and the neuropil of the STG and in neuropil and somata in the commissural ganglia (CGs), brain, and eyestalks. None of the somata within the STG displayed CCKLI. The cross-reactivities of the CCK antisera with several peptides were determined using either a radioimmunoassay or an immunoblot assay; the antisera recognized peptides homologous to CCK but did not cross-react significantly with several unrelated peptides. The STG contains 2 central pattern generators (CPGs), the pyloric and the gastric mill CPGs. Bath application of CCK8 to the STG had modulatory effects on both CPGs, which were dose dependent and reversible. CCK increased the spike frequencies and number of spikes per burst of the pyloric rhythm but had little effect on the period. CCK increased the period of the gastric rhythm and produced changes in the spike frequencies, burst lengths, and phases of gastric units. High concentrations of peptide were needed to produce these effects (10(-6) to 10(-4) M). Finally, stimulation of the stomatogastric nerve (stn), which contains fibers immunoreactive to CCK, produced calcium-dependent release of CCK molar equivalents (CCKE) into the STG. The stn was electrically stimulated and the superfusate around the ganglion was collected and assayed for CCKE using a radioimmunoassay. Stimulation produced the release of 37.1 +/- 7.1 fmol (mean +/- SEM), compared to 13.7 +/- 4.9 fmol for unstimulated controls and 4.9 +/- 2.9 fmol in the absence of calcium. These data suggest that a CCK-like peptide is an endogenous modulator of the stomatogastric ganglion of P. interruptus.