Interactions of suboesophageal ganglion and frontal ganglion motor patterns in the locust

Although locust feeding has been well studied, our understanding of the neural basis of feeding-related motor patterns is still far from complete. This paper focuses on interactions between the pattern of rhythmic movements of the mouth appendages, governed by the suboesophageal ganglion (SOG), and the foregut movements, controlled by the frontal ganglion (FG), in the desert locust. In vitro simultaneous extracellular nerve recordings were made from totally isolated ganglia as well as from fully interconnected SOG-FG and brain-SOG-FG preparations. SOG-confined bath application of the nitric oxide donor, SNP, or the phosphodiesterase antagonist, IBMX, each followed by the muscarinic agonist pilocarpine, consistently induced robust fictive motor patterns in the SOG. This was observed in both isolated and interconnected preparations. In the brain-SOG-FG configuration the SOG-confined modulator application had an indirect excitatory effect on spontaneous FG rhythmic activity. Correlation between fictive motor patterns of the two ganglia was demonstrated by simultaneous changes in burst frequency. These interactions were found to be brain-mediated. Our results indicate the presence of intricate neuromodulation-mediated circuit interactions, even in the absence of sensory inputs. These interactions may be instrumental in generating the complex rhythmic motor patterns of the mandibles and gut muscles during locust feeding or ecdysis-related air swallowing.     

Temporally patterned activity recorded from mandibular nerves of the isolated subesophageal ganglion of Manduca

We recorded bursts of motor neuron activity from closer and opener mandibular nerves of isolated subesophageal ganglia (SOG) and compared them with the feeding motor pattern of intact Manduca larvae. Closer bursts recorded from isolated SOG lasted from 1 to 4s, interburst interval durations lasted from 2 to 49s, and within- and between-animal variability was great. In contrast, motor activity bursts (EMGs) measured from mandibular closer muscles of intact, feeding animals lasted 0.08 to 0.24s with interburst intervals of 0.26 to 0.57s. Variability both within and between animals was small. Bath application of 10(-4)M octopamine to the isolated SOG tended to increase frequency and reduce the duration of bursts, so that they became more like those recorded during feeding.     

Altering cAMP levels within a central pattern generator modifies or disrupts rhythmic motor output

Cyclic AMP is a second messenger that has been implicated in the neuromodulation of rhythmically active motor patterns. Here, we tested whether manipulating cAMP affects swim motor pattern generation in the mollusc, Tritonia diomedea. Inhibiting adenylyl cyclase (AC) with 9-cyclopentyladenine (9-CPA) slowed or stopped the swim motor pattern. Inhibiting phosphodiesterase with 3-isobutyl-1-methylxanthine (IBMX) or applying dibutyryl-cAMP (dB-cAMP) disrupted the swim motor pattern, as did iontophoresing cAMP into the central pattern generator neuron C2. Additionally, during wash-in, IBMX sometimes temporarily produced extended or spontaneous swim motor patterns. Photolysis of caged cAMP in C2 after initiation of the swim motor pattern inhibited subsequent bursting. These results suggest that cAMP levels can dynamically modulate swim motor pattern generation, possibly shaping the output of the central pattern generator on a cycle-by-cycle basis.     

Antennal motor activity induced by pilocarpine in the American cockroach

The antennal motor system is activated by the muscarinic agonist pilocarpine in the American cockroach Periplaneta americana, and its output patterns were examined both in restrained intact animals and in isolated CNS preparations. The three-dimensional antennal movements induced by the hemocoelic drug injection were analyzed in in vivo preparations. Pilocarpine effectively induced prolonged rhythmic movements of both antennae. The antennae tended to describe a spatially patterned trajectory, forming loops or the symbol of infinity (∞). Such spatial regularity is comparable to that during spontaneous tethered-walking. Rhythmic bursting activities of the antennal motor nerves in in vitro preparations were also elicited by bath application of pilocarpine. Cross-correlation analyses of the bursting spike activities revealed significant couplings among certain motor units, implying the spatial regularity of the antennal trajectory. The pilocarpine-induced rhythmic activity of antennal motor nerves was effectively suppressed by the muscarinic antagonist atropine. These results indicate that the activation of the antennal motor system is mediated by muscarinic receptors.     

The locust frontal ganglion: a multi-tasked central pattern generator

The locust frontal ganglion (FG) constitutes a major source of innervation to the foregut dilator muscles and thus plays a key role in control of foregut movements. This paper reviews our recent studies on the generation and characteristics of FG motor outputs in two distinct and fundamental locust behaviors: feeding and molting. In an in vitro preparation, isolated from all descending and sensory inputs, the FG was spontaneously active and generated rhythmic multi-unit bursts of action potentials, which could be recorded from all efferent nerves. Thus the FG motor pattern is generated by a central pattern generator within the ganglion. Intracellular recordings suggest that only a small fraction (10-20%) of the FG 100 neurons demonstrate rhythmic activity. The FG motor output in vivo was relatively complex, and strongly dependent on the locust's physiological and behavioral state. Rhythmic activity of the foregut was found to depend on the amount of food present in the crop; animals with full crop demonstrated higher FG burst frequency than those with empty crop. At the molt, the FG generates a distinct motor pattern that could be related to air-swallowing behavior.     

Descending interneurones from the brain and suboesophageal ganglia and their role in the control of locust behaviour

Lesion and stimulation experiments suggest that the suboesophageal ganglion (SOG) plays a special role in the control of insect behaviour: in bilateral coordination and by maintaining ongoing motor activity. Anatomical observations indicate that there are descending interneurones (DINs) originating in the SOG in addition to those from the brain. An SOG preparation for sampling both types of DIN intracellularly in walking locusts is described. Forty-three units showing activity changes during leg movements and walking were recorded. Using dye injection six were shown to be through-running axons; one was an SOG ascending interneurone; and eight were SOG DINs, 7 contralateral, one ipsilateral. All fired before or during movements and received various sensory inputs. Many gave complex responses to different modalities, several showing directional preferences. Some SOG neurones showed spontaneous changes in activity; activity outlasting movements; or responses to passive as well as active movements. These preliminary results suggest neuronal substrates for the special functions of the SOG in behaviour. They also indicate that DINs, rather than being simple relays, are part of a dynamic network which includes the motor centres. Regulation of complex and subtle aspects of behaviour may be achieved by dynamic and sequential patterns of activity in groups of DINs, some of which may be multifunctional.     

Fictive chewing activity in motor neurons and interneurons of the suboesophageal ganglion of Manduca sexta larvae

Alternating antiphasic rhythmic activity was observed in opener and closer mandibular motor neurons in the isolated suboesophageal ganglion of the larva of Manduca sexta (Lepidoptera: Sphingidae). This was interpreted provisionally as fictive chewing; the pattern is similar to that seen in semiintact animals but of lower frequency. Additionally, a variety of associated rhythmic activities were observed in suboesophageal interneurons. These could be classified into several different physiological types by their activity patterns in relation to the chewing cycle. Some of these neurons can modulate the rhythm when injected with current. It seems likely that they are part of or associated with a central pattern generator circuit for chewing.Abbreviations A anterior - CEC circumoesophageal connective - Cl-MN closer motor neuron - IN interneuron - MdN mandibular nerve - MN motor neuron - O-MN opener motor neuron     

Isolation of functional polarized bile duct units from mouse liver

The development of genetically altered murine animals has generated a need for in vitro systems in the mouse. We have now characterized a novel isolated bile duct unit (IBDU) preparation from the mouse to facilitate such studies. The mouse IBDU is isolated by portal perfusion of collagenase, blunt dissection, further enzymatic digestions, filtering through sized mesh, and culturing on Matrigel for 16-72 h. This mouse IBDU forms a central, enclosed lumen lined by polarized cytokeratin-19-positive cholangiocytes with numerous microvilli on the apical membrane. The IBDU responds to secretory stimuli, including secretin, vasoactive intestinal peptide, IBMX, and forskolin, resulting in expansion of the central lumen from secretion as quantified by videomicroscopy. The secretory response to secretin is dependent on Cl- and HCO3-in the perfusate. These findings indicate that mouse IBDUs are intact, polarized, functional bile duct secretory units that permit quantitative measurements of fluid secretion from mouse bile duct epithelium for the first time. This method should facilitate studies of cholangiocyte secretion in genetically altered murine animal models.     

Extracting temporal relationships between weakly coupled peptidergic and motoneuronal signaling: Application to Drosophila ecdysis behavior

Neuromodulators, such as neuropeptides, can regulate and reconfigure neural circuits to alter their output, affecting in this way animal physiology and behavior. The interplay between the activity of neuronal circuits, their modulation by neuropeptides, and the resulting behavior, is still poorly understood. Here, we present a quantitative framework to study the relationships between the temporal pattern of activity of peptidergic neurons and of motoneurons during Drosophila ecdysis behavior, a highly stereotyped motor sequence that is critical for insect growth. We analyzed, in the time and frequency domains, simultaneous intracellular calcium recordings of peptidergic CCAP (crustacean cardioactive peptide) neurons and motoneurons obtained from isolated central nervous systems throughout fictive ecdysis behavior induced ex vivo by Ecdysis triggering hormone. We found that the activity of both neuronal populations is tightly coupled in a cross-frequency manner, suggesting that CCAP neurons modulate the frequency of motoneuron firing. To explore this idea further, we used a probabilistic logistic model to show that calcium dynamics in CCAP neurons can predict the oscillation of motoneurons, both in a simple model and in a conductance-based model capable of simulating many features of the observed neural dynamics. Finally, we developed an algorithm to quantify the motor behavior observed in videos of pupal ecdysis, and compared their features to the patterns of neuronal calcium activity recorded ex vivo. We found that the motor activity of the intact animal is more regular than the motoneuronal activity recorded from ex vivo preparations during fictive ecdysis behavior; the analysis of the patterns of movement also allowed us to identify a new post-ecdysis phase.     

Sulfated oviductal glycoproteins in the rabbit: quantitation by competitive enzyme-linked immunosorbent assay

The rabbit oviductal epithelium synthesizes and secretes a family of antigenically related, sulfated oviductal glycoproteins (SOG). Anti-SOG monoclonal antibodies (Mabs) were produced and two (Mab 1 and Mab 2) were selected for further characterization. Periodate oxidation of Western blots of oviductal fluid did not affect the binding of Mab 1 or Mab 2, thus suggesting that these antibodies recognized protein rather than carbohydrate epitopes on SOG. The specificity of Mab 1 was determined by Western blot analysis of tissues obtained from estrous rabbits and from the male rabbit reproductive tract. SOG was identified in tissue extracts of both the oviductal ampulla and isthmus. Cervix was the only non-oviductal tissue with which Mab 1 cross-reacted. Mab 1 was used to isolated SOG from whole oviductal fluid by immuno-affinity chromatography. Affinity-purified SOG and Mab 1 were used to develop a quantitative, SOG-specific, competitive enzyme-linked immunosorbent assay. This assay was used to quantify SOG in rabbit oviductal fluid collected during estrus and pseudopregnancy. SOG secretion during pseudopregnancy was resolved into two transient episodes of increased secretion. Maximum SOG secretion (X = 1039 +/- 199 micrograms/day) occurred within 48 h of the induction of pseudopregnancy. A second period of enhanced SOG secretion (X = 308 +/- 46 micrograms/day) occurred during the fifth and sixth days of pseudopregnancy. Baseline SOG secretion occurred during estrus at approximately 60% of maximum postovulatory secretion.     

Differential effects of octopamine and tyramine on the central pattern generator for <Emphasis Type="Italic">Manduca</Emphasis> flight

The biogenic amine, octopamine, modulates a variety of aspects of insect motor behavior, including direct action on the flight central pattern generator. A number of recent studies demonstrate that tyramine, the biological precursor of octopamine, also affects invertebrate locomotor behaviors, including insect flight. However, it is not clear whether the central pattern generating networks are directly affected by both amines, octopamine and tyramine. In this study, we tested whether tyramine affected the central pattern generator for flight in the moth, Manduca sexta. Fictive flight was induced in an isolated ventral nerve cord preparation by bath application of the octopamine agonist, chlordimeform, to test potential effects of tyramine on the flight central pattern generator by pharmacological manipulations. The results demonstrate that octopamine but not tyramine is sufficient to induce fictive flight in the isolated ventral nerve cord. During chlordimeform induced fictive flight, bath application of tyramine selectively increases synaptic drive to depressor motoneurons, increases the number of depressor spikes during each cycle and decreases the depressor phase. Conversely, blocking tyramine receptors selectively reduces depressor motoneuron activity, but does not affect cycle by cycle elevator motoneuron spiking. Therefore, octopamine and tyramine exert distinct effects on the flight central pattern generating network.     

Allatotropic activity in the suboesophageal ganglia and corpora cardiaca of the adult male loreyi leafworm, Mythimna loreyi

Allatotropic activity was found in the methanolic extract of the suboesophageal ganglia (SOG) and the corpora cardiaca (CC) of the Mythimna loreyi virgin males. No allatotropic activity was observed in the extract of brain or corpora allata (CA). Although CA can be activated by the SOG and CC extract, respectively, CC extract inhibited the response to the SOG extract. A significant in vitro allatotropic effect was exerted by the SOG and CC extract within 10 and 15 min, respectively, and this effect can be sustained for several hours even after transferring to fresh medium without extracts. The time course pattern of the CA activation ratio in both the SOG and CC extract-treated group is very similar to, but with significantly higher level than, that in the control group, suggesting the existence of an intrinsic pacemaker or an in vitro effect that controls the fluctuation of the CA biosynthetic activity. Synthetic Manduca sexta allatotropin had no significant effect on the M. loreyi CA. The results of treatment with the adenylate cyclase activator forskolin, the phosphodiesterase inhibitor IBMX, and the cAMP analogue dibutyryl-cAMP did not indicate that cAMP might be involved in the allatotropic control of CA. Arch.     

Role of local nonspiking interneurons in the generation of rhythmic motor activity in the stick insect

Local nonspiking interneurons in the thoracic ganglia of insects are important premotor elements in posture control and locomotion. It was investigated whether these interneurons are involved in the central neuronal circuits generating the oscillatory motor output of the leg muscle system during rhythmic motor activity. Intracellular recordings from premotor nonspiking interneurons were made in the isolated and completely deafferented mesothoracic ganglion of the stick insect in preparations exhibiting rhythmic motor activity induced by the muscarinic agonist pilocarpine. All interneurons investigated provided synaptic drive to one or more motoneuron pools supplying the three proximal leg joints, that is, the thoraco-coxal joint, the coxa-trochanteral joint and the femur-tibia joint. During rhythmicity in 83% (n=67) of the recorded interneurons, three different kinds of synaptic oscillations in membrane potential were observed: (1) Oscillations were closely correlated with the activity of motoneuron pools affected; (2) membrane potential oscillations reflected only certain aspects of motoneuronal rhythmicity; and (3) membrane potential oscillations were correlated mainly with the occurrence of spontaneous recurrent patterns (SRP) of activity in the motoneuron pools. In individual interneurons membrane potential oscillations were associated with phase-dependent changes in the neuron's membrane conductance. Artificial changes in the interneurons' membrane potential strongly influenced motor activity. Injecting current pulses into individual interneurons caused a reset of rhythmicity in motoneurons. Furthermore, current injection into interneurons influenced shape and probability of occurrence for SRPs. Among others, identified nonspiking interneurons that are involved in posture control of leg joints were found to exhibit the above properties. From these results, the following conclusions on the role of nonspiking interneurons in the generation of rhythmic motor activity, and thus potentially also during locomotion, emerge: (1) During rhythmic motor activity most nonspiking interneurons receive strong synaptic drive from central rhythm-generating networks; and (2) individual nonspiking interneurons some of which underlie sensory-motor pathways in posture control, are elements of central neuronal networks that generate alternating activity in antagonistic leg motoneuron pools. © 1995 John Wiley & Sons, Inc.     

A central pattern generator underlies crawling in the medicinal leech

Crawling in the medicinal leech has previously been thought to require sensory feedback because the intact behavior is strongly modulated by sensory feedback and because semi-intact preparations will only crawl if they can move freely. Here we show that an isolated leech nerve cord can produce a crawling motor pattern similar to the one seen in semi-intact preparations, which consists of an anterior-to-posterior wave of alternating excitatory circular and longitudinal motor neuron bursts in each segment. The isolated cord also reproduces the patterns of activity seen in semi-intact preparations for several other kinds of cells: the dorsal inhibitor cell 1, the ventral excitor cell 4, and the annulus erector motor neuron. Because this correspondence is so strong, there must be a central pattern generator in the isolated cord that can produce the basic motor pattern for crawling without sensory feedback. A quantitative analysis of the isolated motor pattern, however, reveals that isolated and semi-intact preparations have longer periods than the intact behavior and that there are deficiencies in the timing of motor neuron bursts in the isolated pattern. These results suggest that sensory feedback modulates the isolated central pattern generator to help produce the normal motor pattern.     

Distinct rhythmic locomotor patterns can be generated by a simple adaptive neural circuit: Biology,simulation, and VLSI implementation

Rhythmic motor patterns can be induced in leg motor neurons of isolated locust thoracic ganglia by bath application of pilocarpine. We observed that the relative phases of levators and depressors differed in the three thoracic ganglia. Assuming that the central pattern generating circuits underlying these three segmental rhythms are probably very similar, we developed a simple model circuit that can produce any one of the three activity patterns and characteristic phase relationships by modifying a single synaptic weight. We show results of a computer simulation of this circuit using the neuronal simulator NeuraLOG/Spike. We built and tested an analog VLSI circuit implementation of this model circuit that exhibits the same range of behaviors as the computer simulation. This multidisciplinary strategy will be useful to explore the dynamics of central pattern generating networks coupled to physical actuators, and ultimately should allow the design of biologically realistic walking robots.     

The brain can eat: Establishing the existence of a central pattern generator for feeding in third instar larvae of Drosophila virilis and Drosophila melanogaster

To establish the existence of a central pattern generator for feeding in the larval central nervous system of two Drosophila species, the gross anatomy of feeding related muscles and their innervation is described, the motor units of the muscles identified and rhythmic motor output recorded from the isolated CNS. The cibarial dilator muscles that mediate food ingestion are innervated by the frontal nerve. Their motor pathway projects from the brain through the antennal nerves, the frontal connectives and the frontal nerve junction. The mouth hook elevator and depressor system is innervated by side branches of the maxillary nerve. The motor units of the two muscle groups differ in amplitude: the elevator is always activated by a small unit, the depressor by a large one. The dorsal protractors span the cephalopharyngeal skeleton and the body wall hence mediating an extension of the CPS. These muscles are innervated by the prothoracic accessory nerve. Rhythmic motor output produced by the isolated central nervous system can simultaneously be recorded from all three nerves. The temporal pattern of the identified motor units resembles the sequence of muscle contractions deduced from natural feeding behavior and is therefore considered as fictive feeding. Phase diagrams show an almost identical fictive feeding pattern is in both species.     

Soldier-specific modification of the mandibular motor neurons in termites

Social insects exhibit a variety of caste-specific behavioral tendencies that constitute the basis of division of labor within the colony. In termites, the soldier caste display distinctive defense behaviors, such as aggressively attacking enemies with well-developed mandibles, while the other castes retreat into the colony without exhibiting any aggressive response. It is thus likely that some form of soldier-specific neuronal modification exists in termites. In this study, the authors compared the brain (cerebral ganglion) and the suboesophageal ganglion (SOG) of soldiers and pseudergates (workers) in the damp-wood termite, Hodotermopsis sjostedti. The size of the SOG was significantly larger in soldiers than in pseudergates, but no difference in brain size was apparent between castes. Furthermore, mandibular nerves were thicker in soldiers than in pseudergates. Retrograde staining revealed that the somata sizes of the mandibular motor neurons (MdMNs) in soldiers were more than twice as large as those of pseudergates. The enlargement of MdMNs was also observed in individuals treated with a juvenile hormone analogue (JHA), indicating that MdMNs become enlarged in response to juvenile hormone (JH) action during soldier differentiation. This enlargement is likely to have two functions: a behavioral function in which soldier termites will be able to defend more effectively through relatively faster and stronger mandibular movements, and a developmental function that associates with the development of soldier-specific mandibular muscle morphogenesis in termite head. The soldier-specific enlargement of mandibular motor neurons was observed in all examined species in five termite families that have different mechanisms of defense, suggesting that such neuronal modification was already present in the common ancestor of termites and is significant for soldier function.     

Inhibition of phosphodiesterases relaxes detrusor smooth muscle via activation of the large-conductance voltage- and Ca²⁺-activated K⁺ channel

Detrusor smooth muscle (DSM) exhibits increased spontaneous phasic contractions under pathophysiological conditions such as detrusor overactivity (DO). Our previous studies showed that activation of cAMP signaling pathways reduces DSM contractility by increasing the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel activity. Here, we tested the hypothesis whether inhibition of phosphodiesterases (PDEs) can reduce guinea pig DSM excitability and contractility by increasing BK channel activity. Utilizing isometric tension recordings of DSM isolated strips and the perforated patch-clamp technique on freshly isolated DSM cells, we examined the mechanism of DSM relaxation induced by PDE inhibition. Inhibition of PDEs by 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor, significantly reduced DSM spontaneous and carbachol-induced contraction amplitude, frequency, duration, muscle force integral, and tone in a concentration-dependent manner. IBMX significantly reduced electrical field stimulation-induced contractions of DSM strips. Blocking BK channels with paxilline diminished the inhibitory effects of IBMX on DSM contractility, indicating a role for BK channels in DSM relaxation mediated by PDE inhibition. IBMX increased the transient BK currents (TBKCs) frequency by ～3-fold without affecting the TBKCs amplitude. IBMX increased the frequency of the spontaneous transient hyperpolarizations by ～2-fold and hyperpolarized the DSM cell resting membrane potential by ～6 mV. Blocking the BK channels with paxilline abolished the IBMX hyperpolarizing effects. Under conditions of blocked Ca(2+) sources for BK channel activation, IBMX did not affect the depolarization-induced steady-state whole cell BK currents. Our data reveal that PDE inhibition with IBMX relaxes guinea pig DSM via TBKCs activation and subsequent DSM cell membrane hyperpolarization.     

Serotonin- and two putative serotonin receptors-like immunohistochemical reactivities in the ground crickets Dianemobius nigrofasciatus and Allonemobius allardi

Serotonin (5-hydroxytryptamine; 5-HT)- and two putative serotonin receptors, 5-HT1A- and 5-HT1B-like, immunohistochemical reactivities were investigated in the cephalic ganglia of two ground crickets, Dianemobius nigrofasciatus and Allonemobius allardi. 5-HT-ir was strongly expressed in the central body, accessory medulla region of the optic lobe, frontal ganglion, posterior cortex of the protocerebrum, dorsolateral region of the protocerebrum, and the suboesphageal ganglion (SOG) in both crickets. However, 5-HT1A-ir and 5-HT1B-ir showed quite mutually distinct patterns that were also distinct from 5-HT-ir. 5-HT1A-ir was located in the pars intercerebralis, dorsolateral region of the protocerebrum, optic tract, optic lobe, and the midline of the SOG in both crickets. 5-HT1B-ir was located in the pars intercerebralis and dorsolateral region of the protocerebrum, and detected weakly in the optic lobe, tritocerebrum, and the midline of the SOG in both crickets. Interspecific differences were observed with 5-HT1A-ir. 5-HT1A-ir was expressed weakly in two neurons in the mandibular neuromere of the SOG in D. nigrofasciatus, while it was expressed strongly in the tritocerebrum, mandibular neuromere, and maxillary neuromere of the SOG in A. allardi and co-localized with CLOCK-ir (CLK-ir). 5HT-1B-ir was co-localized with CLK-ir in the tritocerebrum, mandibular neuromere, and maxillary neuromere of the SOG when double-labeling was conducted in both crickets. These results indicated that 5-HT and both types of 5-HT receptors may regulate circadian photo-entrainment or photoperiodism in A. allardi, while only 5-HT1B may be involved in circadian photo-entrainment or photoperiodism in D. nigrofasciatus.