The Mandibular Ganglion – a new Peripheral Ganglion of the Locust

In addition to the chain of segmental ganglia of the central nervous system (CNS), insects possess a stomatogastric nervous system which innervates the foregut. It consists of the unpaired frontal and hypocerebral ganglia and the paired paraventricular ganglia. It is connected with the central nervous system (CNS) via the frontal connectives, which link the frontal ganglion to both tritocerebral lobes of the supraoesophageal ganglion, or brain (see Fig. 1).

Nerve cells making connections with the frontal ganglion have been found chiefly in the brain, but there are also a few in the suboesophageal ganglion (Aubele and Klemm, 1977; Gundel and Penzlin, 1978; Kirby et al. 1984). Thus, both head ganglia appear to participate in the control of the stomatogastric nervous system.

In the course of a study of the peripheral nervous system of the locust suboesophageal ganglion (Bräunig, 1987), peculiar structures were found in association with one of the major branches of the mandibular nerve. As will be shown here, these structures are hitherto undescribed peripheral ganglia. They contain neurones which project into the suboesophageal ganglion, the tritocere-brum and the stomatogastric nervous system. They may therefore participate in the control of mouthpart and foregut function.

Adult male and female locusts, Locusta migratoria migratorioides (R. & F.) were used for the present study. They were taken from the culture 2–4 days after the final moult, since at that stage fatty tissue around nerves and sense organs is not yet fully developed, greatly facilitating the preparation of delicate components of the peripheral nervous system.

Nerves were stained either by ionophoretically injecting cobalt chloride into the suboesophageal ganglion via a blunt microelectrode, as described previously (Bräunig, 1987), or by the conventional method of exposing single nerve stumps to the staining solution for anterograde or retrograde diffusion. In one set of experiments, the circumoesophageal connectives were stained differentially with cobalt and nickel salts (Quicke and Brace, 1979; Sakai and Yamaguchi, 1983). When necessary, stains within ganglia or small pieces of other tissues were intensified with silver, using the method for wholemounts (Bacon and Altman, 1977).

Since the backfilling method frequently fails to stain all cells of a given population, mandibular ganglia were also stained with Toluidine Blue (Altman and Bell, 1973) to obtain comparative data for cell body numbers. Two mandibular ganglia were embedded in plastic and serially sectioned at 2 μm.

Intracellular staining was carried out using microelectrodes filled with 0.1 mol l⁻¹ hexamminecobaltic chloride (Brogan and Pitman, 1981) and having resistances between 80 and 120 MΩ. The dye was injected for 5–10 min, applying 2–3 nA depolarizing current pulses of 200 ms duration at a frequency of 2.5 Hz.

Nomenclature follows Snodgrass (1928) for muscles and skeletal structures, Altman and Kien (1979) for peripheral nerves of the suboesophageal ganglion and Honomichl (1978a,b) for mandibular mechanoreceptors.

The mandibular ganglion is located about 1mm from the suboesophageal ganglion. It is intricately associated with the mandibular sensory complex, which consists of two muscle receptor organs (DMR, VMR; Honomichl, 1978a,b) and a strand receptor (SR; Honomichl, 1978a). Proximally, the ganglion is attached to the dorsolateral edge of the ventral branch of the mandibular nerve (nerve IB), just where this nerve enters the mandibular hollow. The ganglion tapers towards its dorsal (distal) end, and from it emerge two flattened nerves (PRN, DRN) which connect the ganglion with the tendon of the strand receptor (SR). This tendon is suspended between the posterior margin of the anterior arm of the tentorium and the anterior face of the apodeme of the mandibular closer muscle (M9).

In cobalt-stained material, the mandibular ganglion appears as a group of 22–25 neuronal somata. They either form a cluster close to the lateral edge of nerve IB, or are loosely dispersed along the length of the ganglion between nerve IB and the origin of proximal (PRN) and distal (DRN) strand receptor nerves (Figs 2A and 3). The same number and distribution of cell bodies are observed after Toluidine Blue staining (Fig. 2B).

All cells within the mandibular ganglion are unipolar neurones with elongated somata. Their length ranges from 30 to 50 μm, and their width from 10 to 15 μm. They can be divided into two classes, (i) Four larger cells, located within the distal half of the ganglion, have distally directed neurites (Figs 2A and 3), which proceed into the strand receptor tendon via the distal receptor nerve (DRN). Within this tendon they establish a dense meshwork of dendritic ramifications, from which their axons originate (Fig. 2C). These return towards the mandibular ganglion in the proximal receptor nerve (PRN), bypass other ganglion cells (Figs 2A,C,E and 3), and proceed towards the suboesophageal ganglion in nerve IB. In the following, these neurones will be called strand receptor cells (SR cells), (ii) The second group of cells, named the mandibular ganglion cells (MDG cells), consists of about 20 neurones which are slightly smaller than the SR neurones (Fig. 2A). They usually stain more intensely because their neurites run directly to the suboesophageal ganglion (Fig. 3). Thus, their cell bodies are much closer to the site of dye application (injection of CoCl₂ into the mandibular neuromere of the suboesophageal ganglion) than the SR cell bodies. Two to three MDG cells, always located close to the distal end of the ganglion, have short, distally directed processes (Figs 2E and 3).

The central projections of the SR cells were stained selectively by introducing cobalt chloride into their dendritic tips in the distal end of the receptor tendon (Fig. 4, inset). A maximum of four axons enters the suboesophageal ganglion through the mandibular nerve. Their diameter appears to be rather large, but this is probably only the result of the prolonged silver intensification necessary to show the ramifications within the ganglion (Fig. 4, SOG). Profuse ramifications originate from the axons: they fill the entire ventral mandibular neuropile on the ipsilateral side, so that in wholemounts this neuropile appears to be cupped within a close-meshed basket. Two to three longer collaterals proceed lateroventrally into the maxillary neuromere. Their secondary ramifications are found along the lateral and ventral margin of the maxillary neuropile (Fig. 4, SOG).

One SR fibre makes a U-turn into the circumoesophageal connective. Upon reaching the tritocerebral lobe of the brain, it makes another U-turn into the labrofrontal nerve (Fig. 4, TC) and eventually terminates within the frontal ganglion (Fig. 4, FG). Within the tritocerebrum, there are lateral and ventral areas of ramifications, separated by a major trachea entering the brain.

Of 37 preparations, the tritocerebral ramifications were completely stained in only six cases; those in the frontal ganglion in only one case. This low success rate is due both to the difficult dissection and to the great distances between the ganglia (see Fig. 1).

Attempts were made to stain the single, intersegmentally projecting SR cell by introducing cobalt into its ascending axon either via the frontal or via the circumoesophageal connective. Surprisingly, in all preparations treated in this way, a variable number of MDG cells were stained together with the SR cell (Fig. 2C–E). These MDG cells appear not only within the mandibular ganglion ipsilateral to the filled connective but also in the contralateral ganglion (Fig. 2D, E). This indicates that MDG cells also possess axons which ascend towards the brain and/or the frontal ganglion within the ipsilateral or the contralateral connectives.

For morphological reasons, it is not possible to stain the central projections of MDG cells selectively with the backfilling method. Cutting the MDG and exposing the stump to cobalt stains the MDG cells together with the SR cells and the DMR motor neurone, the axon of which also runs within the proximal receptor nerve (Fig. IB, and Fig. 4, inset). Such preparations, however, confirmed the abovementioned results, in that they showed additional ascending fibres in both circumoesophageal connectives.

To investigate the central projections of MDG cells, attempts were made to penetrate individual cell bodies with microelectrodes. The success rate in these experiments was very low. The cells could not be held for more than a few minutes, a time sufficient only to stain their ramifications within the suboeso-phageal ganglion (Fig. 5), but not those within the tritocerebrum and frontal ganglion. Fig. 5 shows that individual MDG cells project into both ipsi-and contralateral mandibular neuromeres, crossing the sagittal plane of the central nervous system in many places. They also send ascending axons into both circumoesophageal connectives. These features were observed in three successful fills.

Attempts to stain the projections of individual SR cells with intracellular methods failed. Only their neurites within the distal receptor nerve (DRN, inset in Fig. 4) and a few dendritic branches within the receptor strand could be labelled.

To estimate how many MDG cells send axons into both connectives, in one set of experiments one connective was stained with cobalt and the other with nickel. After incubation, mandibular ganglia were exposed to dithiooxamide (rubeanic acid). Precipitation of cobalt and nickel ions with this agent results in complexes of different colours (Quicke and Brace, 1979; Sakai and Yamaguchi, 1983): nickel stains blue, cobalt stains orange, and a mixture of both stains red or purplish. Accordingly, MDG cells with axons in only one connective ought to stain blue or orange, those with axons in both connectives ought to stain red.

Because of the inherent variability of the backfilling method, the number of stained cells in individual mandibular ganglia varied considerably. The total number of stained cells ranged between five and 19 MDG cells (plus one SR cell). However, up to 10MDG cells appeared red or purplish, indicating that at least half the MDG cells of one ganglion project into both connectives. Cells appearing orange or blue might indicate either that individual neurones failed to take up both dyes or the existence of MDG cells with only one ascending axon.

The mandibular ganglion fits a general definition of a ganglion such as that given in Henderson’s Dictionary of Biological Terms; ‘a mass of nerve cell bodies giving rise to nerve fibres’. In contrast to the segmental ganglia of the CNS and the ganglia of the stomatogastric nervous system, however, it contains only cell bodies; there is no neuropile. None of the SR or MDG cells stained by backfilling or by intracellular methods exhibited any side branches within the mandibular ganglion itself (Figs2C – E, 5). Also, serial semithin sections of the ganglia revealed only somata and fibres of passage (neurites and axons of MDG and SR cells, and the motor axon of the DMR).

The neurones of the mandibular ganglia are also clearly different from the peripheral neurosecretory cells found in various insect species (reviewed by Myers and Evans, 1988). The latter are also located on peripheral nerves, their somata in some cases also form small clusters (Fifield and Finlayson, 1978; Garcia-Scheible and Honegger, 1989), but they show ramifications in the immediate vicinity of the cell bodies and appear to lack projections into the central nervous system.

Some previous studies of the peripheral nervous system in other insects make fleeting reference to a ‘swelling of the mandibular nerve’, a ‘mandibular ganglion’ or ‘mandibular association center’ (Haub, 1971; Marquardt, 1940; Rahle, 1970; Swaine, 1920; Zacharuk, 1962), but do not provide any details. Masuko (1986) uses the term ‘ganglionic mass’ for the sensory cells of mandibular mechanoreceptors of the honeybee. It is possible, therefore, that previous authors have seen the MDG cells together with the numerous multipolar sensory cells of the mandibular muscle receptors. In other insects, particularly small species, these are located even closer together than in locusts (beetle: Honomichl, 1978a,b; honeybee: Masuko, 1986). However, as discussed by Moulins (1971), nearby groups of hypopharyngeal sensory neurones have also been mistaken for ganglia. One has to be careful, therefore, with previous interpretations.

In embryos of various orthopteroid insect species, paired peripheral ganglia have been observed in the basal regions of the gnathal appendages (Neumann-Visscher, 1972). Their neurones appear to originate from neuroblasts of the CNS. Stem cells, which do not belong to the segmental sets of neuroblasts that establish the three gnathal ganglionic primordia, have also been observed in locust embryos (Doe and Goodman, 1985). These observations suggest that the neurones of the mandibular ganglia are of central rather than peripheral origin.

“The MDG cells and one of the SR cells (Fig. 4) connect the suboesophageal ganglion with the brain. At least seven MDG cells and the SR cell proceed further into the frontal ganglion (Fig. 2D, E). Thus the neurones connect those neural centres that control the function of the mouthparts and foregut. This suggests that the neurones of the mandibular ganglia are elements of the network involved in the control of feeding. They might participate in the coordination of biting and salivation (suboesophageal ganglion) and swallowing (tritocerebrum and stomato-gastric ganglia). In this sense the mandibular ganglion may be regarded as an additional stomatogastric ganglion.

The mandibular strand receptor was first described in a coleopteran insect (Honomichl, 1978a). Honomichl noted the close proximity of the SR cells to the numerous multipolar sensory neurones of the ventral muscle receptor (VMR, Fig. IB). However, the peculiar morphology of the SR cells was not completely demonstrated, nor were any cells mentioned that might correspond to the locust MDG cells.

It is difficult to compare the mandibular strand receptor with those described for locust legs and antennae (Bräunig and Hustert, 1980; Bräunig, 1985a,b; Pflüger and Burrows, 1987). These have their cell bodies within thoracic ganglia or the brain and do not project to other ganglia, as at least one mandibular SR cell does. Like strand receptors in the thorax (Bräunig, 1985b; Bräunig and Hustert, 1980, 1985) and in the antennae (Bräunig, 1985a), the mandibular strand receptor is a mechanoreceptor which responds to elongation of its receptor strand. Extracellular recordings from the proximal receptor nerve (P. Bräunig, unpublished results) demonstrate units responding to either direct elongation of the strand or elongation caused by upward movement of the closer muscle apodeme. This supports Honomichl’s (1978a) proposed function of the receptor: as a sensor for adduction of the mandible.

The function of the MDG cells remains totally obscure at present in contrast to that of the SR cells. Their cell bodies, for some unknown reason, have been shifted away from the suboesophageal ganglion to form a separate peripheral ganglion together with the SR cells. This displacement of the somata gives rise to the most striking feature of the neurones contained within the mandibular ganglia: the extraordinary length of their neurites (the processes connecting the soma with the nearest dendritic or integrative segment). The MDG cell bodies are 0.7–0.9mm from the CNS, the SR cell bodies are 0.3–0.4mm from the SR tendon. Accordingly, the neurites of both cell types are probably the longest so far encountered within an insect nervous system.

One consequence of this peculiar morphology is that the cell bodies are electrically completely silent. During the intracellular studies, an initial potential drop occurred upon penetration of a cell, but no synaptic potentials or spikes passively invading the cell bodies were observed. To investigate the function OF MDG cells, future studies will have to rely on intracellular recordings from their integrative segments within the suboesophageal ganglion. In view of the small diameters of these processes, this will not be an easy task.

I wish to thank Christine Bohme and Ulrike Paulus for technical assistance. I also wish to thank G. A. Manley for helpful comments on the manuscript and for correcting the English. Finally many thanks go to two unknown referees for their criticism and their extraordinary speed! Supported by Deutsche Forschungs-gemeinschaft (Br 882).