The morphology and fine structure of the giant interneurons of the wood cricket Nemobius sylvestris

The structural and ultrastructural characteristics of giant interneurons in the terminal abdominal ganglion of the cricket Nemobius sylvestris were investigated by means of cobalt and fluorescent dye backfilling and transmission electron microscopy.The projections of the 8 eight pairs of the biggest ascending interneurons (giant interneurons) are described in detail. The somata of all interneurons analyzed are located contralateral to their axons, which project to the posterior region of the terminal ganglion and arborise in the cercal glomerulus. Neuron 7-1a is an exception, because its arborisation is restricted to the anterior region of the ganglion. The fine structure of giant interneurons shows typical features of highly active cells. We observed striking indentations in the perineural layer, enabling the somata of the giant interneurons to be very close to the haemolymph. The cercal glomerulus exhibits a high diversity of synaptic contacts (i.e. axo-dendritic, axo-axonic, dendro-axonic, and dendro-dendritic), as well as areas of tight junctions. Electrical synapses seem to be present, as well as mixed synapses. The anatomical organization of the giant interneurons is finally discussed in terms of functional implications and on a comparative basis.     

Central projections of campaniform sensilla on the cerci of crickets and cockroaches

Summary Campaniform sensilla associated with filiform hairs comprise an important receptor type of the multimodal sensory system of the cerci of crickets and cockroaches. Their axon projections were investigated using iontophoretic cobalt injection into single sensilla.In crickets (Gryllus bimaculatus, Acheta domestica), six different types of cereal campaniform sensilla projections can be distinguished on the basis of their axonal arborizations and terminations. Typically, a proportion of cereal campaniform sensilla, associated with long filiform hairs, give rise to axons that ascend as through fibres from the terminal ganglion to reach the sixth abdominal ganglion. Cereal campaniform sensilla associated with clavate hairs have projections restricted to the terminal ganglion alone.Whereas in crickets axons of cercal campaniform sensilla invade only certain segmental neuropils in the terminal ganglion, in cockroaches (Periplaneta americana) axons from cercal campaniform sensilla branch in every segmental neuropil. A proportion of cereal campaniform sensilla in this species also gives rise to through fibres to the fifth abdominal ganglion.We discuss morphological and functional interpretations of differences between crickets and cockroaches and consider the significance of this type of receptor in the context of previous studies of the cercal system.     

Morphology of soma and dendrites of the giant fiber system in the sixth abdominal ganglion of the cockroach

This study, using the cobalt chloride technique, clarifies the origin of the giant axons in the cockroach, Periplaneta. Each giant axon in the ventral nerve cord arises from a single cell body located in the sixth abdominal ganglion. The position of the soma is always contralateral to the giant axon; it projects anteriorly. In six giant neurons, the axonic and dendritic branches are ipsilateral while the somata are contralateral. In two neurons, both the soma and the dendritic branches are ipsilateral while the axons are contralateral. The dendritic arborizations of the giant neurons form a dense and compact mass of neuropile in each half of the posterior and middorsal part of the ganglion where sensory fibers, primarily from the cercal nerves terminate. The relation of these findings to earlier electrophysiological studies is discussed.     

Morphological and physiological properties of the giant interneuron of the hermit crab (Pagurus pollicaris)

The physiological and morphological properties of the giant interneurons in the hermit crab Pagurus pollicaris are described. The cell bodies are located anteriorly in the supraesophageal ganglion, close to the mid-line. Each cell sends a neurite posteriorly and then laterally, so that they cross over in the center of the ganglion. Each axon then branches: one branch runs laterally while the other travels posteriorly and leaves the ganglion in the circumesophageal connective on the side contralateral to the cell body. The giant axons travel in the circumesophageal connectives and through the thoracic and abdominal ganglia without branching. Each giant axon makes synaptic contact with its ipsilateral giant abdominal flexor motor neuron and with a second flexor motor neuron that has its axon in the contralateral third root. In the supraesophageal ganglion there is a bidirectional synapse between the two giant interneurons. Intracellular recordings from the giant axons show that there is a delay of 0.5 to 0.75 ms that cannot be accounted for by spike propagation along the axons, and may be accounted for by a chemical synapse between the giant interneurons.     

Target neuron specification of short-term synaptic facilitation and depression in the cricket CNS

We investigated the role of retrograde signals in the regulation of short-term synaptic depression and facilitation by characterizing the form of plasticity expressed at novel synapses on four giant interneurons in the cricket cercal sensory system. We induced the formation of novel synapses by transplanting a mesothoracic leg and its associated sensory neurons to the cricket terminal abdominal segment. Axons of ectopic leg sensory neurons regenerated and innervated the host terminal abdominal ganglion forming monosynaptic connections with the medial giant interneuron (MGI), lateral giant interneuron (LGI), and interneurons 7-1a and 9-2a. The plasticity expressed by these synapses was characterized by stimulating a sensory neuron with pairs of stimuli at various frequencies or with trains of 10 stimuli delivered at 100 Hz and measuring the change in excitatory postsynaptic potential amplitude recorded in the postsynaptic neuron. Novel synapses of a leg tactile hair on 7-1a depressed, as did control synapses of cercal sensory neurons on this interneuron. Novel synapses of leg campaniform sensilla (CS) sensory neurons on MGI, like MGI's control synapses, always facilitated. The form of plasticity expressed by novel synapses is thus consistent with that observed at control synapses. Leg CS synapses with 9-2a also facilitated; however, the plasticity expressed by these sensory neurons is dependent on the identity of the postsynaptic cell since the synapses these same sensory neurons formed with LGI always depressed. We conclude that the form of plasticity expressed at these synaptic connections is determined retrogradely by the postsynaptic cell. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 700–714, 1998     

Serotonin immunoreactivity of neurons in the gastropod Aplysia californica

Serotonergic neurons and axons were mapped in the central ganglia of Aplysia californica using antiserotonin antibody on intact ganglia and on serial sections. Immunoreactive axons and processes were present in all ganglia and nerves, and distinct somata were detected in all ganglia except the buccal and pleural ganglia. The cells stained included known serotonergic neurons: the giant cerebral neurons and the RB cells of the abdominal ganglion. The area of the abdominal ganglion where interneurons are located which produce facilitation during the gill withdrawal reflex was carefully examined for antiserotonin immunoreactive neurons. None were found, but two bilaterally symmetric pairs of immunoreactive axons were identified which descend from the contralateral cerebral or pedal ganglion to abdominal ganglion. Because of the continuous proximity of this pair of axons, they could be recognized and traced into the abdominal ganglion neuropil in each preparation. If serotonin is a facilitating transmitter in the abdominal ganglion, these and other antiserotonin immunoreactive axons in the pleuroabdominal connectives may be implicated in this facilitation.     

Cholinergic neurotransmission from mechanosensory afferents to giant interneurons in the terminal abdominal ganglion of the cricket Gryllus bimaculatus

Crickets respond to air currents with quick avoidance behavior. The terminal abdominal ganglion (TAG) has a neuronal circuit for a wind-detection system to elicit this behavior. We investigated neuronal transmission from cercal sensory afferent neurons to ascending giant interneurons (GIs). Pharmacological treatment with 500 muM acetylcholine (ACh) increased neuronal activities of ascending interneurons with cell bodies located in the TAG. The effects of ACh antagonists on the activities of identified GIs were examined. The muscarinic ACh antagonist atropine at 3-mM concentration had no obvious effect on the activities of GIs 10-3, 10-2, or 9-3. On the other hand, a 3-mM concentration of the nicotinic ACh antagonist mecamylamine decreased spike firing of these interneurons. Immunohistochemistry using a polyclonal anti-conjugated acetylcholine antibody revealed the distribution of cholinergic neurons in the TAG. The cercal sensory afferent neurons running through the cercal nerve root showed cholinergic immunoreactivity, and the cholinergic immunoreactive region in the neuropil overlapped with the terminal arborizations of the cercal sensory afferent neurons. Cell bodies in the median region of the TAG also showed cholinergic immunoreactivity. This indicates that not only sensory afferent neurons but also other neurons that have cell bodies in the TAG could use ACh as a neurotransmitter.     

Transplantation of neurons reveals processing areas and rules for synaptic connectivity in the cricket nervous system

In order to assess the nature of spatial cues in determining the characteristic projection sites of sensory neurons in the CNS, we have transplanted sensory neurons of the cricket Acheta domesticus to ectopic locations. Thoracic campaniform sensilla (CS) function as proprioceptors and project to an intermediate layer of neuropil in thoracic ganglia while cercal CS transduce tactile information and project into a ventral layer in the terminal abdominal ganglion (TAG). When transplanted to ectopic locations, these afferents retain their modality-specific projection in the host ganglion and terminate in the layer of neuropil homologous to that of their ganglion of origin. Thus, thoracic CS neurons project to intermediate neuropil when transplanted to the abdomen and cercal CS neurons project to a ventral layer of neuropil when transplanted to the thorax. We conclude that CS can be separated into two classes based on their characteristic axonal projections within each segmental ganglion. We also found that the sensory neurons innervating tactile hairs project to ventral neuropil in any ganglion they encounter after transplantation. Ectopic sensory neurons can form functional synaptic connections with identified interneurons located within the host ganglia. The new contacts formed by these ectopic sensory neurons can be with normal targets, which arborize within the same layer of neuropil in each segmental ganglion, or with novel targets, which lack dendrites in the normal ganglion and are thus normally unavailable for synaptogenesis. These observations suggest that a limited set of molecular markers are utilized for cell–cell recognition in each segmentally homologous ganglion. Regenerating sensory neurons can recognize novel postsynaptic neurons if they have dendrites in the appropriate layer of neuropil. We suggest that spatial constraints produced by the segmentation and the modality-specific layering of the nervous system have a pivotal role in determining synaptic specificity. © 1993 John Wiley & Sons, Inc.     

Differential backfilling of interneuron populations based upon axon projections in a lobster abdominal ganglion

Intersegmental interneurons in the lobster second abdominal ganglion were differentially stained by simultaneously backfilling their axons in the anterior and posterior hemiconnectives with nickel and cobalt ions. When precipitated with rubeanic acid the nickel and cobalt formed different colors in the cell bodies depending upon the location of the axon(s) in the connectives. In the most completely stained preparations 55 ascending, 56 descending, and 25 bidirectional interneurons were observed. Soma diameters ranged from 15-120 micron. One of the somata was located in the connective. The differential staining technique has advantages over conventional backfilling techniques for examining the morphological relationships of populations of neurons having different axon projections.     

Multiple feedback loops in the flying cockroach: excitation of the dorsal and inhibition of the ventral giant interneurons

1. In a tethered cockroach (Periplaneta americana) whose wings have been cut back to stumps, it is possible to elicit brief sequences of flight-like activity by puffing wind on the animal's body. 2. During such brief sequences, rhythmic bursts of action potentials coming from the thorax at the wingbeat frequency, descend the abdominal nerve cord to the last abdominal ganglion (A6). This descending rhythm is often accompanied by an ascending rhythm (Fig. 2). 3. Intracellular recording during flight-like activity from identified ascending giant interneurons, and from some unidentified descending axons in the abdominal nerve cord, shows that: (a) ventral giant interneurons (vGIs) remain silent (Fig. 3); (b) dorsal giant interneurons (dGIs) are activated at the onset of the flight-like activity and remain active sporadically throughout the flight sequence (Fig.4); (c) some descending axons in the abdominal nerve cord show rhythmic activity phase-locked to the flight rhythm (Fig. 5). 4. Also during such brief sequences, the cercal nerves, running from the cerci (paired, posterior, wind sensitive appendages) to the last abdominal ganglion, show rhythmic activity at the wingbeat frequency (Fig. 6). This includes activity of some motor axons controlling vibratory cercal movements and of some sensory axons. 5. More prolonged flight sequences were elicited in cockroaches whose wings were not cut and which flew in front of a wind tunnel (Fig. 1B). 6. In these more prolonged flight sequences, the number of ascending spikes per burst was greater than that seen in the wingless preparation (Fig. 8; compare to Fig. 2). Recordings from both ventral and dorsal GIs show that: in spite of the ongoing wind from both the tunnel and the beating wings, which is far above threshold for the vGIs in a resting cockroach, the vGIs are entirely silent during flight. Moreover, the vGIs response to strong wind puffs that normally evoke maximal GI responses is reduced by a mean of 86% during flight (Fig. 9). The dGIs are active in a strong rhythm (Figs. 11 and 12). 7. Three sources appear to contribute to the ascending dGI rhythm (1) the axons carrying the rhythmic descending bursts; (2) the rhythmic sensory activity resulting from the cercal vibration; and (3) the sensory activity resulting from rhythmic wind gusts produced by the wingbeat and detected by the cerci. The contribution of each source has been tested alone while removing the other two (Figs. 13 and 14). Such experiments suggest that all 3 feedback loops are involved in rhythmically exciting the dGIs (Fig. 15).     

Pretreatment of the cockroach cercal afferent/giant interneuron synapses with nicotinoids and neonicotinoids differently affects acetylcholine and nicotine-induced ganglionic depolarizations

We have recently demonstrated that neonicotinoid insecticides were able to act as agonists of postsynaptic nicotinic acetylcholine receptors (nAChRs) expressed at the synapse between the cercal nerve XI and the giant interneurons, in the sixth abdominal ganglion. In this work, we demonstrated that nicotinoids such as nornicotine acted as an agonist of nicotinic acetylcholine receptors expressed at cercal afferent/giant interneurons while cotinine was a poor agonist. Indeed, nornicotine induced a ganglionic depolarization which was blocked by the nicotinic antagonist mecamylamine. In addition, we found that pretreatment of the sixth abdominal ganglion with 1 and 10 μM nornicotine and cotinine had no significant effect on acetylcholine and nicotine-induced depolarization. But pretreatment with 1 and 10 μM acetamiprid and imidacloprid had a strong effect. 1 and 10 μM acetamiprid completely blocked acetylcholine-induced depolarization, whereas imidacloprid had a partial effect. The present work therefore suggests, in agreement with previous studies, that nornicotine and cotinine bind to distinct cockroach postsynaptic nAChRs, whereas acetamiprid and imidacloprid have competitive effects with acetylcholine and nicotine on ganglionic depolarization.     

Synapse formation by sensory neurons after cross-species transplantation in crickets: the role of positional information

The role of positional information in synapse formation was studied in the cricket cercal sensory system by transplanting epidermis from one species of cricket to another. Strips of cercal epidermis containing identified sensory neurons were transplanted from a black donor species to a tan host species; the color difference was used to distinguish between donor and host tissue in adults. Transplanted sensory neurons regenerated axons into the host terminal abdominal ganglion where they formed functional chimeric synapses. These methods were used to test the role of positional information in central synapse formation. Newly generated sensory neurons, formed by the donor tissue at the border between graft and host, were examined to test the idea that their position would determine their structure, function, and projection pattern. These "intercalated" sensory neurons support the positional information hypothesis. First, they had directional sensitivities which were appropriate to their location on the cercus; receptors of this directionality would never be made by the donor tissue if left in its original position. Second, these sensory neurons projected to regions of the CNS known to be appropriate for their directionality. Finally, simultaneous recordings from these ectopic sensory neurons and host interneurons demonstrated the expected synaptic connection, based on the overlap of pre- and postsynaptic cells. Thus three aspects of receptor function, directionality, afferent projection, and choice of synaptic partners, appeared to be controlled by positional information.     

Cercal sensory regulation of acetylcholinesterase in nervous system of the cockroach, Periplaneta americana

Cercal ablation caused a significant loss in acetylcholinesterase (AChE) activity of the cercal nerves and terminal ganglion within 12 hr while a similar reduction in enzyme activity of connectives was noticed at least one day after cercectomy. The decrease in AChE activity of the nervous tissues showed a recovery toward control levels from 20 days of unilateral cercectomy whereas the bilateral cercectomy produced a continuous and irreversible decline in enzyme activity. These localized changes in AChE activity of the abdominal nervous system of the cockroach were attributed to be regulated by the cercal sensory innervation.     

Regeneration of cercal filiform hair sensory neurons in the first-instar cockroach restores escape behavior

Neural regeneration in the escape circuit of the first-instar cockroach is described using behavioral analysis, electrophysiology, intracellular staining, and electron microscopy. Each of the two filiform hairs on each of the animal's cerci is innervated by a single sensory neuron, which specifically synapses with a set of giant interneurons (GIs) in the terminal ganglion. These trigger a directed escape run. Severing the sensory axons causes them to degenerate and perturbs escape behavior, which is restored to near normal after 4–6 days. Within this time, afferents regenerate and reestablish arborizations in the terminal ganglion. In most cases, regenerating afferents enter the cercal glomerulus and re-form most of the specific monosynaptic connections they acquired during embryogenesis, although their morphology deviates markedly from normal; these animals reestablish near normal escape behavior. In a few cases, regenerating afferents remain within the cercus or bypass the cercal glomerulus, and thereby fail to re-form synapses with GIs; these animals continue to exhibit perturbed escape behavior. We conclude that in most cases, specific synapses are reestablished and appropriate escape behavior is restored. This regeneration system therefore provides a tractable model for the establishment of synaptic specificity in a simple neuronal circuit. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 439–458, 1997     

Immunocytochemical localization of nicotinic acetylcholine receptors in the terminal abdominal ganglion of the cockroach (Periplaneta americana)

A polyclonal, monospecific antiserum raised against a nicotinic acetylcholine receptor protein affinity-purified from insect nervous tissue, was employed to demonstrate the localization of antigenic sites in the neuropile of the terminal (sixth) abdominal ganglion of the cockroach Periplaneta americana. In agreement with previously published autoradiographic mapping of specific [125I]alpha-bungarotoxin binding sites, specific areas of the central neuropile of this ganglion were densely stained, but not the cercal afferent axons. No staining was detected corresponding to the dense, peripheral, partly non-specific binding of alpha-bungarotoxin seen in autoradiographs of the same tissue. Certain peripherally located neuronal cell bodies, including the cell body of giant interneuron 2, contained intracellularly located antigenic sites.     

Neuromodulation of flight initiation by octopamine in the cockroach Periplaneta americana

We have tested the effect of a known insect neuromodulator, octopamine, on flight initiation in the cockroach. Using minimally dissected animals, we found that octopamine lowered the threshold for windevoked initiation of flight when applied to either of two major synaptic sites in the flight circuitry: 1) the last abdominal ganglion, where wind-sensitive neurons from the cerci excite dorsal giant interneurons, or 2) the metathoracic ganglion, where the dorsal giant interneurons activate interneurons and motoneurons which are involved in producing the rhythmic flight motor pattern in the flight muscles (Fig. 2).Correlated with this change in flight initiation threshold, we found that octopamine applied to the last abdominal ganglion increased the number of action potentials produced by individual dorsal giant interneurons when recruiting the cereal wind-sensitive neurons with wind puffs (Figs. 3, 4, 5) or with extracellular stimulation of their axons (Fig. 6). Octopamine increases the excitability of the giant interneurons (Figs. 7, 8). Also, when we stimulated individual dorsal giant interneurons intracellularly, the number of action potentials needed to initiate flight was reduced when octopamine was applied to the metathoracic ganglion (Fig. 9).Abbreviations EMG electromyogram - dGIs dorsal giant interneurons - GI giant interneuron - A6 sixth abdominal ganglion - T3 third thoracic ganglion - EPSP excitatory postsynaptic potential     

Distribution of glutamate decarboxylase-like immunoreactivity in the sixth abdominal ganglion of the cockroach Periplaneta americana

Summary An antiserum against glutamate decarboxylase (GAD) of the rat brain was used to locate GAD activity in sections of the nervous system of the cockroach, Periplaneta americana. The sixth abdominal ganglion was chosen because electrophysiological evidence suggests the presence of GABAergic inhibitory synapses in the cereal-giant interneuron system. Groups of somata and numerous fibres and tracts were positively labelled by the GAD antiserum. A posterior group of labelled somata could be identified close to the entry of the cereal nerves. A line of somata clusters lay along a ventro-lateral furrow. Another discrete row of GAD-like cells was located dorso-laterally. Some small cells among the dorsal unpaired neurons were labelled. A small central group appeared under these cells. An abundance of GAD-like processes and transversal tracts were found within the neuropile. The different systems of GABAergic inhibitors in the ganglion are discussed; in particular we show that the fibres of cereal nerve X are not labelled. This demonstrates that the latter act on the giant fibres via interneurons. We suggest that the group that sends axons into the overlapping region between the cereal nerve and the giant fibre could be the inhibitory interneurons involved in this system.     

Serotonin-like immunoreactivity in the ventral nerve cord of the Colorado potato beetle,Leptinotarsa decemlineata: Identification of five different neuron classes

Summary In an immunohistochemical study of the ventral nerve cord of L. decemlineata, five distinct neuron categories were distinguished: 1) Two paired segmental twin interneurons occur in each ganglion or neuromere; their axons distribute processes over almost the entire nerve cord and run to the cerebral ganglion complex. In contrast, other axons are distributed locally. 2) Four large frontal neurosecretory neurons occur in the suboesophageal ganglion (SOG), two of which have axons that run into the mandibular nerves to form a neurohemal plexus on the surface of cerebral nerves. 3) A pair of large caudal neurons occur in the terminal ganglion and innervate the hindgut. 4) Local miniature interneurons occur in the SOG. 5) Terminal neurons are present in the last abdominal ganglion. Segmental twin interneurons appear to be grouped into 3 functional units spanning several ganglia. Their axons run to specific projection areas, which separate the functional units, and which mark the externally visible separation of condensed ganglion complexes. A possible role of the most caudal functional unit might be the synaptic control of caudal neurons innervating the hindgut.     

Parasitoid wasp sting: A cocktail of GABA,taurine, and β‐alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host

The wasp Ampulex compressa injects venom directly into the prothoracic ganglion of its cockroach host to induce a transient paralysis of the front legs. To identify the biochemical basis for this paralysis, we separated venom components according to molecular size and tested fractions for inhibition of synaptic transmission at the cockroach cercal‐giant synapse. Only fractions in the low molecular weight range (<2 kDa) caused synaptic block. Dabsylation of venom components and analysis by HPLC and MALDI‐TOF‐MS revealed high levels of GABA (25 mM), and its receptor agonists β‐alanine (18 mM), and taurine (9 mM) in the active fractions. Each component produces transient block of synaptic transmission at the cercal‐giant synapse and block of efferent motor output from the prothoracic ganglion, which mimics effects produced by injection of whole venom. Whole venom evokes picrotoxin‐sensitive chloride currents in cockroach central neurons, consistent with a GABAergic action. Together these data demonstrate that Ampulex utilizes GABAergic chloride channel activation as a strategy for central synaptic block to induce transient and focal leg paralysis in its host. © 2006 Wiley Periodicals, Inc. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006