Divergent co-transmitter actions underlie motor pattern activation by a modulatory projection neuron

Co-transmission is a common means of neuronal communication, but its consequences for neuronal signaling within a defined neuronal circuit remain unknown in most systems. We are addressing this issue in the crab stomatogastric nervous system by characterizing how the identified modulatory commissural neuron (MCN)1 uses its co-transmitters to activate the gastric mill (chewing) rhythm in the stomatogastric ganglion (STG). MCN1 contains gamma-aminobutyric acid (GABA) plus the peptides proctolin and Cancer borealis tachykinin-related peptide Ia (CabTRP Ia), which it co-releases during the retractor phase of the gastric mill rhythm to influence both retractor and protractor neurons. By focally applying each MCN1 co-transmitter and pharmacologically manipulating each co-transmitter action during MCN1 stimulation, we found that MCN1 has divergent co-transmitter actions on the gastric mill central pattern generator (CPG), which includes the neurons lateral gastric (LG) and interneuron 1 (Int1), plus the STG terminals of MCN1 (MCN1(STG)). MCN1 used only CabTRP Ia to influence LG, while it used only GABA to influence Int1 and the contralateral MCN1(STG). These MCN1 actions caused a slow excitation of LG, a fast excitation of Int1 and a fast inhibition of MCN1(STG). MCN1-released proctolin had no direct influence on the gastric mill CPG, although it likely indirectly regulates this CPG via its influence on the pyloric rhythm. MCN1 appeared to have no ionotropic actions on the gastric mill follower motor neurons, but it did use proctolin and/or CabTRP Ia to excite them. Thus, a modulatory projection neuron can elicit rhythmic motor activity by using distinct co-transmitters, with different time courses of action, to simultaneously influence different CPG neurons.     

Proctolin activates an inward current whose voltage dependence is modified by extracellular Ca2+.

The pentapeptide proctolin modulates the activity of the rhythmic pattern generators in the crustacean stomatogastric nervous system. Proctolin strongly excites the lateral pyloric and the inferior cardiac neurons of the stomatogastric ganglion (STG), causing them to fire extended high-frequency bursts of action potentials (Hooper and Marder, 1987; Nusbaum and Marder, 1989a,b). We now report that proctolin depolarizes these cells maximally at membrane potentials close to the threshold for action potential generation. In voltage clamp, proctolin evokes an inward current, carried at least partially by Na+, that shows strong outward rectification. Removal of extracellular Ca2+ markedly increases the amplitude of the proctolin-evoked current and linearizes its current-voltage curve. The properties of the proctolin current make it ideally suited to contribute to the activity-dependent modulation of the pyloric network of the STG.     

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.     

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.     

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.     

Orcokinin peptides in developing and adult crustacean stomatogastric nervous systems and pericardial organs

The orcokinins are a family of neuropeptides recently isolated from several crustacean species. We found orcokinin-like immunoreactivity in the stomatogastric nervous systems and pericardial organs of three decapod crustacean species, Homarus americanus, Cancer borealis, and Panulirus interruptus. The neuropil of the stomatogastric ganglion was stained in adults of all three species as well as in embryonic and larval H. americanus. In H. americanus, the somata giving rise to this projection were found in the inferior ventricular nerve. Matrix-assisted laser desorption/ionization mass spectrometry mass profiling and sequencing with postsource decay led to the identification of six different orcokinin family peptides, including those previously described in other decapods and two novel shorter peptides. Application of exogenous [Ala(13)]orcokinin to the stomatogastric ganglion of H. americanus resulted in changes in the pyloric rhythm. Specifically, the number of lateral pyloric (LP) neuron spikes/burst decreased, and the phase of firing of the pyloric neurons was altered. Together, these data indicate that the orcokinins are likely to function as modulators of the crustacean stomatogastric ganglion.     

Modulatory action and distribution of the neuropeptide proctolin in the crustacean stomatogastric nervous system

Immunocytochemical methods were used to map the distribution of proctolinlike immunoreactivity in the stomatogastric nervous systems (stomatogastric ganglion (STG), paired commissural ganglia (CG), oesophageal ganglion (OG), and connecting nerves) of three crustacean species: Panulirus interruptus, Cancer borealis, and Homarus americanus. Although the patterns of proctolinlike staining were similar among the three species, some differences were also observed. Over 70% of the proctolinlike material in STGs, as measured by radioimmunoassay, was indistinguishable from authentic proctolin in reverse-phase high-performance liquid chromatography. Bath application of proctolin to STGs from Cancer and Panulirus induced characteristic and robust (though somewhat different) changes in their motor patterns. The threshold concentration was approximately 10(-9)M proctolin, and the effects were dose-dependent. These data suggest that the neuropeptide proctolin serves as a neuromodulator of the stomatogastric ganglion.     

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.     

Modulation of the lobster pyloric rhythm by the peptide proctolin

The modulation of the pyloric network of the stomatogastric ganglion (STG) of the lobster Panulirus interruptus by the neuropeptide proctolin is described. First, the effects of proctolin on the pyloric motor patterns were characterized in terms of frequency and phase relations. Pyloric cycle frequency and lateral pyloric (LP) neuron activity increased and ventricular dilator (VD) neuron activity decreased with increasing concentrations (10(-9)-10(-6) M) of applied proctolin. Next, the effects of proctolin on the individual neurons that constitute the pyloric network were determined. Identified neurons were isolated from chemical and electrical presynaptic inputs by using pharmacological agents (Marder and Eisen, 1984a) and/or photoinactivation following Lucifer yellow injection (Miller and Selverston, 1979). Proctolin increased the amplitude and frequency of bursts produced by isolated pacemaker anterior burster (AB) neurons. Isolated LP and pyloric (PY) neurons responded to proctolin with increases in activity only when they were at or above threshold. All other pyloric neurons were unaffected. To determine how the direct effects of proctolin on isolated neurons resulted in the observed changes in frequency and phase relations in the motor pattern of the intact pyloric circuit seen in proctolin, individual neurons were deleted from the circuit. A comparison of proctolin's effects on isolated neurons with those on the intact network shows that the synaptic connectivity among neurons directly affected by proctolin and those unaffected by it shapes the network's response to proctolin.     

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.     

Forskolin reduces a transient potassium current in lobster neurons by a cAMP-independent mechanism

Forskolin decreases the transient potassium current, IA, in voltage-clamped somata of identified neurons in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. The diterpene reduces the peak outward current and accelerates the rate of inactivation of IA. Forskolin has no detectable effects on two other identifiable potassium currents in these cells, IK(Ca) and IK(V). Three identified stomatogastric neuron types (PD, PY, AB) have marked amounts of IA which are affected by forskolin; three other cell types (LP, IC, VD) have little or no IA, and forskolin has no effect on their outward currents. Bath application of 8-bromo-cAMP, N,N-dibutyryl-cAMP and IBMX do not affect IA. In addition, the forskolin analog, 1,9-dideoxyforskolin, which does not activate adenylate cyclase, mimics forskolin's effects on IA. Thus, the effects of forskolin on IA are not mediated by cAMP elevation.     

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.     

Identification and distribution of a proctolin-like neuropeptide in the nervous system of the gypsy moth, Lymantria dispar, and in other Lepidoptera

Although the neuropeptide proctolin has important functions in many arthropods, it is reported to be absent in Lepidoptera. Its possible occurrence in these insects was reinvestigated by bioassays of HPLC fractions and immunocytochemistry. A proctolin-like substance was recovered from the frontal and subesophageal ganglia of Lymantria dispar. This substance has the same chromatographic retention time as proctolin; enzymatic degradation indicates that it is a peptide; it is bound by proctolin antisera; and thus it is indistinguishable from authentic proctolin. A small subpopulation of proctolin-like immunoreactive (PLI) neurons was stained in the larval CNS of L. dispar, Manduca sexta, Trichoplusia ni, Galleria mellonella, and Vanessa cardui. Most prominent of these cells are median neurosecretory neurons in the brain, paired neurons in the frontal ganglion, two clusters of neurons in the subesophageal ganglion, paired lateral neurons in the thoracic ganglia, and dorsomedial neurons in the abdominal ganglia. Also, varicose PLI axons are found in the corpora cardiaca and perivisceral organs. In L. dispar, PLI cells also were found in the corpora cardiaca. The results of this study indicate that proctolin is of general occurrence in the Lepidoptera, that it has an important role in the stomatogastric nervous system, and that it may be released as a local neurohormone from various neurohemal organs.     

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.     

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.     

The voltage-dependent, slow inward current induced by the neuropeptide FMRFamide in Aplysia neuron R14

The effects of the peptide FMRFamide (Phe-Met-Arg-Phe-NH2) on the soma of neuron R14 in the abdominal ganglion of Aplysia californica and A. brasiliana were characterized. Pressure-ejected FMRFamide caused 3 types of responses, (1) a fast outward current (duration, less than 30 sec), (2) a fast inward current (duration, less than 20 sec), and (3) a slow inward current (peak at 0.5-1 min; duration, 2-3 min). The slow inward current, the chief object of this study, arises from a voltage-dependent conductance increase. The FMRFamide-elicited slow inward current is largest between -40 mV and -20 mV, the region of a negative slope resistance in the normal current-voltage relationship for R14. The slow FMRFamide-induced inward current is largely carried by Na+. This current is independent of external [K+] but depends inversely on external [Ca2+] and [Cl-]. The concentrations of the latter ions may influence the voltage dependence of the response. The slow inward current has many properties in common with inward currents induced in other molluscan neurons by applications of neuropeptides or intracellular injections of cyclic nucleotides.     

Neuromodulatory complement of the pericardial organs in the embryonic lobster,Homarus americanus

The pericardial organs (POs) are a pair of neurosecretory organs that surround the crustacean heart and release neuromodulators into the hemolymph. In adult crustaceans, the POs are known to contain a wide array of peptide and amine modulators. However, little is known about the modulatory content of POs early in development. We characterize the morphology and modulatory content of pericardial organs in the embryonic lobster, Homarus americanus. The POs are well developed by midway through embryonic (E50) life and contain a wide array of neuromodulatory substances. Immunoreactivities to orcokinin, extended FLRFamide peptides, tyrosine hydroxylase, proctolin, allatostatin, serotonin, Cancer borealis tachykinin-related peptide, cholecystokinin, and crustacean cardioactive peptide are present in the POs by approximately midway through embryonic life. There are two classes of projection patterns to the POs. Immunoreactivities to orcokinin, extended FLRFamide peptides, and tyrosine hydroxylase project solely from the subesophageal ganglion (SEG), whereas the remaining modulators project from the SEG as well as from the thoracic ganglia. Double-labeling experiments with a subset of modulators did not reveal any colocalized peptides in the POs. These results suggest that the POs could be a major source of neuromodulators early in development.     

Distribution and partial characterization of FMRFamide-like peptides in the stomatogastric nervous systems of the rock crab, Cancer borealis, and the spiny lobster, Panulirus interruptus

The distribution of FMRFamide-like peptides was studied in the complete stomatogastric nervous system [the paired commissural ganglia, single oesophageal ganglion, and the single stomatogastric ganglion (STG)] of two decapod crustacean species, the spiny lobster Panulirus interruptus and the rock crab Cancer borealis, by using immunocytochemical techniques. Antiserum 231 from the O'Donohue laboratory and antiserum 671C (described here) gave essentially the same staining patterns. In the commissural ganglia of both species there were ten to 20 stained neurons and dense neuropilar staining. The oesophageal ganglion of the crab had four stained neurons. Lucifer Yellow backfills followed by immunostaining showed that the two larger stained neurons of the oesophageal ganglion sent processes into the inferior ventricular nerve. The two smaller neurons sent processes into the inferior oesophageal nerves. The oesophageal ganglion of the lobster had two stained neurons that sent processes into the inferior ventricular nerve as well. None of the somata of the STG stained in either species, but in both species stained fibers were seen in the stomatogastric nerve that entered the STGs and ramified profusely throughout the neuropil. In some preparations of the crab, a stained fiber was visible in the dorsal ventricular nerve. The amounts of the FMRFamide-like peptides found in all regions of the nervous system of P. interruptus and C. borealis were determined by radioimmune assay (RIA). Column chromatography and high-performance liquid chromatography suggest that, in both species, much if not all of the RIA-assayable material is accountable for by peptides that are larger and more hydrophobic than FMRFamide.     

Distribution of modulatory inputs to the stomatogastric ganglion of the crab, Cancer borealis.

The pyloric and gastric mill neural networks in the crustacean stomatogastric ganglion receive modulatory inputs from more anteriorly located ganglia via the stomatogastric nerve. In this study we employed biocytin backfilling and immunostaining, as well as electron microscopy, to determine the origin of these inputs in the crab, Cancer borealis. Fiber counts from electron micrographs of sections through the stomatogastric nerve showed that this nerve contains 55-60 medium to large diameter fibers (1-13 microns). These fibers were individually wrapped by several layers of membrane, presumably glial in origin. There was also a single cluster of jointly wrapped, small diameter (< 1 micron) fibers that may originate from peripheral sensory somata. Biocytin backfills revealed that approximately two thirds of the individually wrapped fibers in this nerve originate from somata in the other three ganglia of the stomatogastric nervous system, including the paired commissural ganglia and the single oesophageal ganglion. There were approximately 20 biocytin-labeled somata in each commissural ganglion and 3 somata in the oesophageal ganglion. An additional ten somata were localized to the stomatogastric ganglion itself. This accounts for nearly all of the medium to large diameter fibers in the stomatogastric nerve. We used double-labeling with backfills and immunocytochemistry to determine that there are two proctolin-immunoreactive neurons and four FMRFamide-like immunoreactive neurons among the biocytin-labeled neurons in each commissural ganglion. Both peptides modulate neural network activity in the stomatogastric ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage clamp analysis of intact stomatogastric neurons

Two-electrode voltage clamp of intact, identified pyloric neurons of the spiny lobster stomatogastric ganglion reveals two major outward currents. A rapidly inactivating, tetraethylammonium- (TEA) insensitive, 4-aminopyridine- (4AP) sensitive, outward current resembles IA of molluscan neurons; it activates rapidly on depolarizations above rest (e.g. -45 mV), delaying both the axonal-sodium and the neuropil-calcium spikes which escape voltage-clamp control. We infer that A-current is distributed both in a space clamped region (on or near the soma) and in a non-space clamped region with access to the generators for sodium and calcium spikes. A calcium-dependent outward current, IO(Ca), activates rapidly at clamp steps above -25 mV and inactivates at depolarizing holding voltages. Increasing depolarization results in an increase in both IO(Ca) and firing rate but a reduction in the amplitude of the sodium spike current. Blockage of IO(Ca) with Cd2+ causes little change in spike firing pattern. These observations are consistent with IO(Ca) being activated primarily in the soma and nearby regions which are under good control with a soma voltage clamp (and distant from the Na(+)-spike trigger zone). While the lack of space clamp limits resolution of charging transients and tail currents, the identification of the major current subgroups can still be readily accomplished, and inferences about the location and function of currents can be made which would not be possible if the cells were space clamped or truncated.