Neuroanatomy and neurophysiology of the locust hypocerebral ganglion

The insect stomatogastric ganglia control foregut movements. Most previous work on the system has concentrated on the frontal ganglion (FG), including research into the role of the FG in feeding as well as molting-related behavior, mostly in locusts, but also in other insect species. The stomatogastric system exerts its physiological actions by way of careful interaction and coordination between its different neural centers and pattern-generating circuits. One such hitherto unstudied neural center is the hypocerebral ganglion (HG), which is connected to the FG via the recurrent nerve. It sends two pairs of nerves along the esophagus and to the posterior region of the crop, terminating in the paired ingluvial ganglia. Very little is known about the neuroanatomy and neurophysiology of the insect HG. Here we investigate, for the first time, the neuronal composition of the locust HG, as well as its motor output. We identify rhythmic patterns endogenous to the isolated HG, demonstrating the presence of a central pattern-generating network. Our findings suggest interactions between the HG and FG rhythm-generating circuits leading to complex physiological actions of both ganglia. This work will serve as a basis for future investigation into the physiology of the HG and its role in insect behavior.     

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.     

The role of the frontal ganglion in foregut movements of the moth,Manduca sexta

Two types of rhythmic foregut movements are described in fifth instar larvae of the moth, Manduca sexta. These consist of posteriorly-directed waves of peristalsis which move food toward the midgut, and synchronous constrictions of the esophageal region, which appear to retain food within the crop. We describe these movements and the muscles of the foregut that generate them.The firing patterns of a subset of these muscles, including a constrictor and dilator pair from both the esophageal and buccal regions of the foregut, are described for both types of foregut movement.The motor patterns for the foregut muscles require innervation by the frontal ganglion (FG), which lies anterior to the brain and contains about 35 neurons. Eliminating the ventral nerve cord, leaving the brain and FG intact, did not affect the muscle firing patterns in most cases. Eliminating both the brain and the ventral nerve cord, leaving only the FG to innervate the foregut, generally resulted in an increased period for both gut movements and muscle bursts. This manipulation also produced increases in burst durations for most muscles, and had variable effects on the phasing of muscle activity. Despite these changes, the foregut muscles still maintained a rhythmic firing pattern when innervated by the FG alone.Two nerves exit the FG to innervate the foregut musculature: the anteriorly-projecting frontal nerve, and the posteriorly-directed recurrent nerve. Cutting the frontal nerve immediately and irreversibly stopped all muscle activity in the buccal region, while cutting the recurrent nerve immediately stopped all muscle activity in the pharyngeal and esophageal regions. Recordings from the cut nerves leaving the FG showed that the ganglion was spontaneously active, with rhythmic activity continuing within the nerves. These observations indicate that all of the foregut muscle motoneurons are located within the FG, and the FG in isolation produces a rhythmic firing pattern in the motoneurons. We have identified several motoneurons within the FG, by cobalt backfills and/or simultaneous intracellular recordings and fills from putative motoneurons and their muscles.Abbreviations BC Buccal Constrictor - BC1 buccal constrictor motoneuron 1 - BC2 buccal constrictor motoneuron 2 - BD Buccal Dilator - BD1 buccal dilator motoneuron 1 - EC Esophageal Dilator - EC1 esophageal dilator motoneuron 1 - EC2 esophageal dilator motoneuron 2 - EC3 esophageal dilator motoneuron 3 - ejp excitatory junction potential - FG frontal ganglion - psp postsynaptic potential     

Feeding-related motor patterns of the locust suboesophageal ganglion induced by pilocarpine and IBMX

The mandibular motor pattern induced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) in isolated locust suboesophageal ganglia (SOG) was investigated and compared with the motor pattern induced by pilocarpine in an already established preparation of the SOG. Motor patterns occurring after bath application of IBMX or pilocarpine were recorded extracellularly from suitable nerves of isolated SOG. For a quantitative evaluation of long (15 min) sequences of rhythmic neural activity containing several hundred cycles, spectral analysis of spike trains was applied. Using a set of characteristic parameters extracted from spectra computed for each individual preparation, quantitative comparisons of the rhythms induced by IBMX and pilocarpine were made. Significant differences in regularity, frequency of oscillation, and intra-burst frequency were found whereas the phase relationships of different motor pools were similar. Differences in the effect of the drugs on the activity recorded extracellularly from mandibular closer motoneurones were investigated further using intracellular recordings. Our findings imply that the IBMX-induced motor pattern is a suitable in vitro model of mandibular central motor control like the pilocarpine induced pattern. The better regularity is an advantageous feature for further experiments on central pattern generation. Information on second messengers involved in central pattern generation provided by the pharmacological profile of IBMX forms a basis for pharmacological and histological investigations on the mandibular central pattern generating network.     

Mechanoresponsive neurones in the suboesophageal ganglion of the locust

Abstract. A preparation is described for intracellular recording from the neur-opile of the sub-oesophageal ganglion (SOG) of the locust, while stimulating the labial and maxillary palps with plant material in such a way as to mimic the palpation behaviour which precedes and continues throughout feeding. Twelve neurones responding to simulated palpation were recorded from and stained in the SOG. Axons of three neurones ascended to the brain, six had descending axons and three had all of their processes confined to the suboesophageal ganglion. The major regions of arborization were in the ventrolateral and mediolateral neuropiles of the maxillary and labial neuromeres. All twelve neurones were solely mechanoresponsive. In addition to responding to palpation of one or more of the four palps, five also responded to stimulation of the labrum, one to touching each antenna, and one to mechanical stimulation of each of the six tarsi. In the context of what is known about the role of mechano-stimulation in the control of feeding, and given their particular patterns of input and arborizations, it is suggested that the neurones may be active during food selection and ingestion.     

Flight motor patterns recorded in surgically isolated sections of the ventral nerve cord ofLocusta migratoria

Summary In the locust,Locusta migratoria, the pairs of connectives between the three thoracic ganglia and in the neck were transected in all possible combinations. Each of these preparations was tested for the production of rhythmic flight motor activity, with sensory input from the wing receptors intact and after deafferentation. The motor activity elicited in these preparations was characterized by intracellular recordings from motoneurons and electromyographic analyses.The motor patterns observed in locusts with either the neck or the pro-mesothoracic connectives severed (Figs. 2, 3, and 4) were very similar to the flight motor pattern produced by animals with intact connectives. The activity recorded in mesothoracic flight motoneurons of locusts with either only the meso-metathoracic connectives cut or both the meso-metathoracic and the neck connectives transected were similar to each other. Rhythmic motor activity could be observed in these preparations only as long as sensory feedback from the wing receptors was intact. These patterns were significantly different from the intact motor pattern (Figs. 5, 6, and 7). Similar results were obtained when the mesothoracic ganglion was isolated from the other two thoracic ganglia, although the oscillations produced under these conditions were weak (Fig. 8 upper). In the isolated metathorax no rhythmic flight motor activity could be recorded (Fig. 8 lower), even when wing afferents were intact.Considering the differences between the motor patterns observed in the various preparations these results suggest that the ganglia of the locust ventral nerve cord do not contain segmental, homologous flight oscillators which are coupled to produce the intact flight rhythm. Instead they support the idea that the functional flight oscillator network is distributed throughout the thoracic ganglia (Robertson and Pearson 1984). The results also provide further evidence that sensory feedback from the wing sense organs is necessary for establishing the correct motor pattern in the intact animal (Wendler 1974, 1983; Pearson 1985; Wolf and Pearson 1987 a).Abbreviations CPG central pattern generator - EMG electromyogram     

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.     

Neuromodulation for behavior in the locust frontal ganglion

Neuromodulators orchestrate complex behavioral routines by their multiple and combined effects on the nervous system. In the desert locust, Schistocerca gregaria, frontal ganglion neurons innervate foregut dilator muscles and play a key role in the control of foregut motor patterns. To further investigate the role of the frontal ganglion in locust behavior, we currently focus on the frontal ganglion central pattern generator as a target for neuromodulation. Application of octopamine, a well-studied insect neuromodulator, generated reversible disruption of frontal ganglion rhythmic activity. The threshold for the modulatory effects of octopamine was 10^–6 mol l^–1, and 10^–4 mol l^–1 always abolished the ongoing rhythm. In contrast to this straightforward modulation, allatostatin, previously reported to be a myoinhibitor of insect gut muscles, showed complex, tri-modal, dose-dependent effects on frontal ganglion rhythmic pattern. Using a novel cross-correlation analysis technique, we show that different allatostatin concentrations have very different effects not only on cycle period but also on temporal characteristics of the rhythmic bursts of action potentials. Allatostatin also altered the frontal ganglion rhythm in vivo. The analysis technique we introduce may be instrumental in the study of not fully characterized neural circuits and their modulation. The physiological significance of our results and the role of the modulators in locust behavior are discussed.Abbreviation CPG central pattern generator - FG frontal ganglion - JH juvenile hormone - STNS stomatogastric nervous system     

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.     

Properties of descending dorsal unpaired median (DUM) neurons of the locust suboesophageal ganglion

A group of six dorsal unpaired median (DUM) neurons of the suboesophageal ganglion (SOG) of locusts was studied with neuroanatomical and electrophysiological techniques. The neurons are located posteriorly in the SOG and have axons that descend into the ganglia of the ventral nerve cord, some as far as the terminal abdominal ganglion. Within thoracic ganglia the neurons have profuse dendritic ramifications in many neuropiles, including ventral sensory neuropiles. Based on their projection patterns three different morphological types of neurons can be distinguished. These neurons receive excitatory inputs through sensory pathways that ascend from the thoracic ganglia and are activated by limb movements. They may be involved in the modulation of synaptic transmission in thoracic ganglia.     

Proprioceptive reflex interactions with central motor rhythms in the isolated thoracic ganglion of the shore crab

Summary Experiments were carried out on an isolated central nervous system preparation of the shore crab,Carcinus maenas, comprising the fused thoracic ganglion complex with two proprioceptors of one back leg still attached. These, the thoracic-coxal muscle receptor organ and the coxo-basal chordotonal organ, monitor movement and position of the first and second joints, respectively. Motor activity was recorded extracellularly from the central cut ends of the nerves innervating the promotor and remotor muscles of the thoracic-coxal joint, and the levator and depressor muscles of the coxal-basal joint of the same leg. Simultaneous intracellular recordings were made from central processes of individual motoneurones of each muscle.In the absence of any sensory input, the isolated ganglion exhibited rhythmic bursting in the motor nerve roots, with a slow, usually irregular cycle period of 5–50 s.Both receptor organs had both intra-joint and inter-joint effects on the rhythmically active preparation. In most cases the coxo-basal receptor organ had the greater effect.Resistance reflexes initiated by each of the joint proprioceptors were modulated by the rhythmic activity.It may be concluded that, while the isolated thoracic ganglion of the crab is capable of generating rhythmic motor output, proprioceptive feedback from the two basal joints is important in shaping the motor patterns underlying locomotion. Inappropriate reflexes which would impede active movements about these joints are modulated or reversed so as to permit and even reinforce intended locomotory movements.     

Phase-dependent coordination of two motor programs in the buccal ganglion of a pteropod mollusk

Rhythmic activities of two feeding structures of the pteropod mollusk Clione limacina, redula and hooks, controlled by the neural networks in the buccal ganglia must be coordinated in order to produce a meaningful feeding response. Optical recording from the buccal ganglia, which allows the simultaneous activities of numerous neurons to be traced, revealed that such coordination exists in a phase-dependent manner. Instead of recording four theoretically possible phases of neuronal rhythmic activity, we always recorded only two phases, even after the electrical stimulation of the cerebro-buccal connective, which triggers both radula and hook rhythmic movements in the preparation.     

Cholinergic activation of the lobster cardiac ganglion

The frequency of rhythmic burst activity of the isolated lobster cardiac ganglion is increased by exogenously applied acetylcholine and muscarinic agonists. Responses of individual motor neurons isolated from the ganglion by transection consist of a slow depolarization and repetitive bursting. The pharmacological profile of the receptors mediating this response is similar to that of vertebrate neuronal muscarinic receptors. Isolated ganglia incubated in the presence of [3H]-choline (18-19 h) exhibited radiolabelled acetylcholine accumulation. It is suggested that ganglionic excitation may be accomplished by extrinsic or intrinsic activation of muscarinic receptors on the motor neurons.     

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     

Descending unpaired median neurons with bilaterally symmetrical axons in the suboesophageal ganglion of Manduca sexta larvae

Three large median cell bodies with a diameter between 40 and 70 μm that exhibit octopamine immunoreactivity were identified in the posterior part of the suboesophageal ganglion of the tobacco hawkmoth larva, Manduca sexta. These neurons possess bilaterally symmetrical axons in the posterior neck connectives, and at least one of them extends through the whole ventral nerve cord to the terminal abdominal ganglion. Therefore, these neurons belong to the class of descending ventral unpaired median neurons. From each cell body, a primary neurite ascends anteriorly, which after bending dorsally turns posteriorly and then bifurcates to give rise to two descending axons. From the primary neurite two main dendritic branches ascend anteriorly, and four characteristic branches can be distinguished originating from them: two descending dendritic branches and two ascending dendritic branches. Dense arborizations from all these branches exist in all neuromeres of the suboesophageal ganglion. Intracellular recordings from these neurons show that in contrast to the ventral unpaired median neurons of thoracic and abdominal ganglia, they do not produce overshooting action potentials but exhibit passive soma spikes only. During pharmacologically evoked fictive motor patterns these neurons show coupling to various motor patterns such as crawling, feeding and molting.     

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.     

Generation of specific behaviors in a locust by local release into neuropil of the natural neuromodulator octopamine

The natural insect neuromodulator octopamine (OCT) was released iontophoretically into regions of neuropil in locust metathoracic ganglia. A narrowly-defined site was found on one side of the ganglion at which release caused a prolonged bout of repetitive flex-extend-flex movements of the tibia on the injected side, at a frequency of from 2-3.5 Hz. When a bout had terminated, repetition of the OCT release caused an extremely similar bout to occur, and again with further treatments, indefinitely. OCT iontophoresis at the equivalent site on the contralateral side caused the contralateral flexor to make stepping movements. Two sites were found, in each half of the ganglion, at which similar OCT release evoked a bout of flight motor activity at 10 Hz. The flight bout involved both sides synchronously and nearly equally, except for a slightly greater motor output on the injected side. Evoked bouts lasted from 20 sec to 25 min depending on the preparation and amount of OCT released. At a site in the 6th abdominal ganglion of mature female locusts OCT release suppressed ongoing rhythmic oviposition digging evoked by severing the ventral nerve cord. A number of previously undescribed DUM neurons was encountered and their dendritic patterns, which are distinctive, determined following dye injection. A hypothesis, termed the Orchestration Hypothesis is presented, which considers how modulator neurons such as locust octopaminergic neurons, might be involved in the generation of specific behaviors.     

Evidence for homologous peptidergic neurons in the buccal ganglia of diverse nudibranch mollusks.

The buccal ganglia of seven nudibranches (Aeolidia papillosa, Armina californica, Dirona albolineata, D. picta, Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea) were examined to explore possible homologies between large cells that reacted with antibodies directed against small cardioactive peptide B (SCPB). The buccal ganglion of each species possessed a pair of large, dorsal-lateral, whitish neurons that contained an SCPB-like peptide. We refer to these neurons as the SLB (SCPB-immunoreactive Large Buccal) cells. In all species examined, the SLB cells project out the gastroesophageal nerves and appear to innervate the esophagus. In each species, an apparent rhythmic feeding motor program (FMP) was observed by intracellular recording from both SLB neurons and other neurons in isolated preparations of the buccal ganglia. SLB cells often fire at a high frequency, and usually burst in a specific phase relation to the FMP activity. Stimulation of SLB cells enhances expression of the feeding motor program, either by potentiating existing activity or eliciting the FMP in quiescent preparations. Finally, perfusion of isolated buccal ganglia with SCPB excites the SLB cells and activates FMPs. Thus, both the immunohistochemical and electrophysiological data suggest that the SLB cells within three suborders of the opisthobranchia (Dendronotacea, Arminacea, and Aeolidacea) are homologous. A comparison of our data with previously published studies indicates that SLB cell homologs may exist in other gastropods as well.