Insect Central Nervous System Research Articles

Bombyxin gene expression in tissues other than brain detected by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization.

Bombyxin is a 5 kDa insulin-related peptide produced in four pairs of medial neurosecretory cells in the brain of the silkmoth Bombyx mori. We demonstrate here the presence of bombyxin mRNA in tissues other than brain: ganglia, epidermis, testis, ovary, fat body, silk gland, Malpighian tubule, midgut, and hindgut of the Bombyx fifth instar larvae. Bombyxin mRNA was detected by Oligotex reverse transcription-polymerase chain reaction (RT-PCR), a rapid and simple procedure of reverse transcription-PCR, and in situ hybridization. The Oligotex RT-PCR method effectively eliminated the contaminating DNA in RNA samples and amplified bombyxin mRNA efficiently. In situ hybridization of the Bombyx ovary clearly demonstrated the localization of the bombyxin mRNA in the ovariole. The present study is the first demonstration of expression of brain neurosecretory peptide in tissues other than the central nervous system in insects at RNA level.

Neurogenesis in the insect central nervous system

Recent advances provide an increasingly sophisticated understanding of Drosophila CNS development. First, genes have been identified that specify unique neuroblast cell fates. Second, neuroblasts have been found to use a novel mechanism for asymmetric localization of proteins into one daughter cell at mitosis. Third, a gene controlling the choice between glial/neuronal determination has been discovered. And finally, new cell-lineage methods are being used to determine the entire lineage of identified neuroblasts, including axon projections and synaptic contacts.

Morphological and electrophysiological aspects of dorsal median paired neurons generating plateau action potentials in the terminal abdominal ganglion of the insect central nervous system

Among the three clusters of dorsal unpaired median neurons of the Periplaneta americana terminal abdominal ganglion, another type of neuron has been characterized by anterograde cobalt stainings and microelectrode technique. These neurons are bilaterally distributed in the ganglion. Their axons ipsilaterally exit the ganglion via the anterior proctodeal nerves, to innervate the proctodeum. They are characterized by a long-duration overshooting action potentials and a low firing frequency. Most often the depolarizing phase is composed of two peaks: a fast spike followed by a slow phase. Tetrodotoxin suppressed the fast peak and blocked the spontaneous activity suggesting that sodium channels are involved in the depolarizing phase as well as in the initiation of the action potential. Calcium channel blockers induced a disappearing of the slow depolarizing phase indicating the participation of calcium ions and a reduction of the afterhyperpolarization reflecting the participation of calcium-activated potassium channels. Furthermore, cadmium, as lanthanum or barium, induced a long-lasting plateau potential, which would be due to a persistent sodium conductance. Tetraethylammonium increased the duration of the action potential indicating that potassium channels are implicated in the falling phase. The results demonstrate that these neurons are different from other cells, especially dorsal unpaired median neurons, of the central nervous system of the cockroach.

Immunocytochemistry of a novel GABA receptor subunitRdl inDrosophila melanogaster

Following our recent cloning of a novel gamma-aminobutyric acid (GABA) receptor subunit gene Resistance to dieldrin or Rdl form cyclodiene resistance locus in Drosophila melanogaster, we were interested in defining its pattern of expression during development. Here we report the raising of an anti-Rdl polyclonal antibody that recognizes a single protein of the expected 65 kDa size in immunoblots of Drosophila head homogenates. In situ hybridization using Rdl cDNA probes and the anti-Rdl antibody shows that Rdl message and protein are highly expressed in the developing central nervous system (CNS) of 15-17 h embryos. Interestingly, despite the use of GABA in both the peripheral and CNS of insects, Rdl GABA receptor subunits appear to be confined to the CNS. Detailed immunocytochemistry of Drosophila brain sections showed particularly strong anti-Rdl antibody staining in the optic lobes, ellipsoid body, fan shaped body, ventrolateral protocerebrum and the glomeruli of the antennal lobes. Results are compared with the distribution of staining observed in the insect CNS with antibodies against GABA itself and synaptotagmin, a synaptic vesicle protein.

Paroxysmal activity increased in subjects under habitual eating of MSG

Cellular and organismal iron storage depends on the function of the ferritin protein complex in insects and mammals alike. In the central nervous system of insects, the distribution and relevance of ferritin remain unclear, though ferritin has been implicated in Drosophila models of Alzheimers' and Parkinsons' disease and in Aluminum-induced neurodegeneration. Here we show that transgene-derived expression of ferritin subunits in glial cells of Drosophila melanogaster causes a late-onset behavioral decline, characterized by loss of circadian rhythms in constant darkness and impairment of elicited locomotor responses. Anatomical analysis of the affected brains revealed crystalline inclusions of iron-loaded ferritin in a subpopulation of glial cells but not significant neurodegeneration. Although transgene-induced glial ferritin expression was well tolerated throughout development and in young flies, it turned disadvantageous at older age. The flies we characterize in this report contribute to the study of ferritin in the Drosophila brain and can be used to assess the contribution of glial iron metabolism in neurodegenerative models of disease.

Immunocytochemical localization of sodium channels in an insect central nervous system using a site-directed antibody.

Antibodies to channel proteins and specific peptide sequences have been previously used to localize voltage-activated sodium channels in the rat brain. Here we describe the first localization of sodium channels in an insect nervous system using a site-directed antibody. The mesothoracic ganglion of the cockroach was stained with an antibody to the highly conserved SP19 sequence. Antibody labelling was visualized by light microscopy using the avidin/biotin method on wax sections, and transmission electron microscopy of immunogold-labelled thin sections. Central ganglia of insects contain clearly separated regions of cell bodies, synaptic neuropil, axon tracts, and nerves. Antibody staining by light microscopy was limited to neurons, and was intense in axons throughout the ganglion and nerves. Staining was also strong in the cytoplasm, but not the nuclei, of many neuronal cell bodies. Neuropil regions were relatively lightly labelled. These findings can be correlated with the known electrophysiology of the ganglion. Electron microscopy detected sodium channels in areas surrounding axons, probably including axon membranes and enveloping glial cell membranes. Axonal mitochondria were also heavily labelled, suggesting a sodium channel transport function for these organelles.

Regenerating Adult Cockroach Dorsal Unpaired Median Neurones In Vitro Retain their In Vivo Membrane Characteristics

ABSTRACT The ability of differentiated neurones to recover from disease or injury depends upon both intrinsic and extrinsic factors. Whereas most mammalian neurones have a limited capacity for regeneration, regulated, in part, by physical and chemical cues in the brain microenvironment (Bray et al. 1987; Caroni and Schwab, 1988, 1989), invertebrates, and in particular insects, exhibit a far greater capacity for repair of central neurones and circuits (Treherne et al. 1988). Studies of the cues that regulate the regenerative process are made easier by the use of individual, identified neurones, cultured under controlled conditions. Invertebrates are particularly useful in this regard; neurones from mature nervous systems of both annelids and molluscs have been grown successfully in culture and their growth can be influenced by changes in the culture conditions (Acklin and Nicholls, 1990; Dagan and Levitan, 1981; Ready and Nicholls, 1979; Syed et al. 1990). Routine and long-term culture of identified neurones from the insect central nervous system (CNS) has proved more elusive, preventing the use of neurones from these well-studied systems. Recently, however, cultures of cockroach (Howes et al. 1991), locust (Kirchoff and Bicker, 1992) and moth (Hayashi and Levine, 1992) adult neurones have been described.

A blood-derived attachment factor enhances the in vitro growth of two glial cell types from adult cockroach.

Reactive glial cells from chemically-lesioned areas of the central nervous system (CNS) of adult cockroaches (Periplaneta americana) have been grown in vitro on a substrate of fibronectin. This paper reports the enhancement of growth that is achieved when blood cells, serum, or medium conditioned by a 2-h incubation with blood cells are used as an alternative substrate. Glial cells rapidly grew out from connective explants to form extensive radial mats of cells linking up with those from adjacent explants on each of the blood-derived substrates. In addition to supporting the growth of reactive glial cells, characterised by their long, thin, branching morphology, these substrates also revealed the presence of a second type of glial cell, not previously found on fibronectin. Such cells, derived from ganglionic explants, behaved in a very different way to the reactive glia, initially spreading out to form a flattened sheet of phase-bright cells, before migrating away over the culture surface. The growth-enhancing effects of this blood-derived factor may play a role in the events following damage to the insect CNS, where it is known that the entry of blood cells into the lesion site is an important precursor to the rapid and structured repair seen in this system.

Optical recording of neuronal activity in the insect central nervous system: odorant coding by the antennal lobes of honeybees.

Voltage-sensitive dyes and activity-dependent intrinsic optical signals were used to study the spatio-temporal activity in the antennal lobes of honeybees. The intrinsic signals are somewhat slower than the dye signals but show a 10-fold larger intensity change. These intrinsic signals consist of at least two components--one is wavelength-independent and the other strongly wavelength-dependent, with a maximum at approximately 500 nm. Local inhibitory connections within the antennal lobes were examined by recording the activity elicited by an electrical stimulus to the antennal nerve of a slice preparation before and after applying picrotoxin to manipulate GABAergic inhibitory synapses. The inhibition starts with a delay of approximately 10 ms after onset of the response and has at least two components. The spatial distribution of the inhibition is extremely inhomogeneous, with areas of small inhibition adjacent to areas of large inhibition. Thus inhibitory interactions in the antennal lobes are not evenly distributed among the glomerular organization. Stimulation of an in vivo preparation with an odour yields a spatially restricted activity. However, the spatial map appears highly dynamic in time because the size of the activated area is a function of the time during and after the stimulus.

A novel kainate receptor in the insect nervous system

The pharmacology of excitatory amino acid receptors on an identified neurone in the insect central nervous system was investigated using single electrode recording techniques. Application of kainate, domoate and quisqualate elicited large, slow depolarizations with a rise time of approximately 4 min and recovery time of 23 min. Concentration-response curves were constructed giving an order of potency, domoate > quisqualate > kainate, and fitted curves demonstrated quisqualate to be a possible partial agonist, compared to domoate and kainate. Agonist responses were insensitive to the antagonists CNQX and picrotoxin and the vertebrate receptor-subtype selective agonists AMPA and trans-ACPD did not elicit any response, suggesting a novel type of excitatory amino acid receptor present on neurones in the insect central nervous system.

Origins of the insect enteric nervous system: differentiation of the enteric ganglia from a neurogenic epithelium

The enteric nervous system (ENS) of the moth Manduca sexta is organized into two distinct cellular domains: an anterior domain that includes several small ganglia on the surface of the foregut, and a more posterior domain consisting of a branching nerve plexus (the enteric plexus) that spans the foregut-midgut boundary. Previously, we showed that the neurons of the posterior domain, the enteric plexus, are generated from a large placode that invaginates from the caudal lip of the foregut; subsequently, the cells become distributed throughout the enteric plexus by a sequence of active migration. We now demonstrate that the neurons of the anterior domain, the cells of the enteric ganglia, arise via a distinct developmental sequence. Shortly after the foregut has begun to form, three neurogenic zones differentiate within the foregut epithelium and give rise to chains of cells that emerge onto the foregut surface. The three zones are not sites of active mitosis, as indicated by the absence of labelling with a thymidine analogue and by clonal analyses using intracellularly injected dyes. Rather, the zones serve as loci through which epithelial cells are recruited into a sequence of delamination and neuronal differentiation. As they emerge from the epithelium, the cells briefly become mitotically active, each cell dividing once or twice. In this manner, they resemble the midline precursor class of neural progenitors in the insect central nervous system more than neuroblast stem cells. The progeny of these zone-derived precursors then gradually coalesce into the ganglia and nerves of the anterior ENS. Although this reorganization results in some variability in the precise configuration of neurons within the ganglia, the overall morphology of the ganglia is highly stereotyped, consisting of cortical layers of cells that surround a ventral neuropil. In addition, a number of the neurons within the frontal and hypocerebral ganglia express identifiable phenotypes in a manner that is similar to many cells of the insect central nervous system. These observations indicate that the differentiation of the enteric ganglia in Manduca involves an unusual combination of features seen during the formation of other regions of the nervous system and, as such, constitutes a distinct program of neurogenesis.

Electrophysiological activity of the C-peptide of the Locusta insulin-related peptide Effect on the membrane conductance of Locusta neurones in vitro

The C-peptide of Locusta insulin-related peptide, which is a 50 residue peptide originally isolated from the corpora cardiaca of the insect Locusta migratoria and to which we refer as 5-kDa peptide, has been synthesised chemically by the solid-phase metho, using a BOC strategy. Since this peptide contains in its sequence a potential monobasic cleavage site, we also synthesised its 1–38 residue-related fragment, named 4-kDa peptide, although we have no hints of its natural occurrence in the corpora cardiaca. Electrophysiological studies have shown that both the 5-kDa and 4-kDa peptides depolarise the membrane and increase the membrane conductance of neurones freshly isolated from the thoracic ganglia of Locusta. Under voltage-clamp conditions, the current underlying these effects was inwardly directed and could be resolved into 2 components. One component, I(5-kDa) 1, activated at potentials more hyperpolarised than −50 mV, peaked at about −75 mV and was blocked by the potassium channel blockers cesium and rubidium. The second component, I(5-kDa) 2 was activated at potentials more depolarised than −50 mV, increased with depolarisation and was not blocked by cesium and rubidium. The effects of the 5-kDa and 4-kDa peptides on the membrane potential and membrane conductance of Locusta neurones suggest that these peptides may have a physiological role in the central nervous system of insects.

Insect neural evolution—a fugue or an opera?

Insects are exceedingly diverse, including more species than all other animal groups combined. The burden of this essay is that, in spite of the wondrous variation in external body form, the central nervous systems of insects are remarkably conservative, varying astonishingly little from the most primitive to the most advanced of the extant groups. Instead of evolutionary variation commensurate with that of the body, we see tinkering with the connections formed by a largely conserved set of neurons.

Neural repair and glial proliferation: Parallels with gliogenesis in insects

There is a growing recognition, stemming from work with both vertebrates and invertebrates, that the capacity for neuronal regeneration is critically dependent on the local microenvironment. That environment is largely created by the non-neuronal elements of the nervous system, the neuroglia. Therefore an understanding of how glial cells respond to injury is crucial to understanding neuronal regeneration. Here we examine the process of repair in a relatively simple nervous system, that of the insect, in which it is possible to define precisely the cellular events of the repair process. This repair is rapid and well organised; it involves the recruitment of blood cells, the division of endogenous glial elements and, possibly, migration from pre-existing glial pools in adjacent ganglia. There are clear parallels between the events of repair and those of normal glial development. It seems likely that the ability of the insect central nervous system to repair resides in the retention of developmental capacities throughout its life and that damage results in the activation of this potential.

Pharmacological characterization and chemical fractionation of the venom of the ponerine ant, Paraponera clavata (F.)

1. The neurotoxic action of the venom of the ponerine ant, Paraponera clavata, was studied using a cascade of mammalian smooth muscle preparations and a preparation for investigating transmission from fibres of the cercal nerve to a giant interneuron in the sixth abdominal ganglion of the cockroach. 2. The venom contains three toxic fractions that block synaptic transmission in the insect central nervous system. 3. Two of these fractions have agonistic action on mammalian smooth muscle preparations. 4. One of the later fractions was characterized pharmacologically as containing a kinin. 5. The other, and most active neurotoxic fraction, was rechromatographed, resulting in the purification of a peptide of 25 amino acids residues, called poneratoxin, PoTX: Phe-Leu-Pro-Leu-Leu-Ile-Leu-Gly-Ser-Leu-Leu-Met-Thr-Pro-Pro-Val-Ile-Gln-Ala-Ile-His-Asp-Ala-Gln-Arg-HN 2.

Regional Variation in the Current Flow Across an Insect Blood–Brain Barrier

One notable characteristic of the insect and vertebrate central nervous system is the presence of a clearly defined blood-brain barrier. In the insect this barrier is made up of the perineurial glial cells, and shows a heterogeneity in structure and possibly function between the connectives and ganglia. In this paper we have used a two-dimensional vibrating probe to investigate the net flow of electrical current across the barrier at various locations in the abdominal nervous system. The results show clear differences between different areas. There is a strong and consistent outward current flow (3.16 microA cm-2) perpendicular to the ganglion surface over the equatorial plane. Current returns through the peripheral nerves and the connectives. A detailed study of the latter shows that the net inward flow is principally over the initial section, immediately adjacent to the ganglia (2.11 microA cm-2). The different current polarities can be correlated with structural differences in the underlying glial cells.

Sodium channel polypeptides in central nervous systems of various insects identified with site directed antibodies

Immunoprecipitation, radiophosphorylation and SDS-PAGE autoradiography enable the characterization of sodium channel polypeptides in the central nervous system of insects belonging to four phylogenetically distinct orders: grasshoppers, cockroaches, flies and moth larvae. It has been shown that the insect sodium channels: (1) Are recognized by the previously described (Gordon et al. (1988) Biochemistry 27, 7032–7038) site directed antibodies corresponding to a highly conserved segment linking the homologous domains III and IV in the vertebrate sodium channel α subunits. (2) Serve as substrates for phosphorylation by cAMP-dependent protein kinase. (3) Are devoid of disulfide linkage to smaller subunits unlike sodium channels in vertebrate brain. (4) Are glycoproteins as shown in the grasshopper by the decrease of apparent molecular weight following endoglycosidase F treatment and specific binding to the lectins concanavalin A and wheat germ agglutinin. (5) Reveal a diversity with regard to their (a) apparent molecular masses which range from 240 to 280 kDa and (b) V8 proteinase digestion phosphopeptides indicating either differences in the positioning of the enzymatic cleavage and/or phosphorylation sites. These results provide the first evidence for structural diversity of sodium channel subtypes among various insect orders and are compared to their mammalian counterparts.

Cholinergic Nerve Terminals in the Central Nervous System of Insects

Cholinergic Nerve Terminals in the Central Nervous System of Insects

Blocking actions of heptachlor at an insect central nervous system GABA receptor

The actions of the polychlorocycloalkane insecticide heptachlor, and its epoxide metabolite, were examined on GABA receptors in insects and vertebrates. Electrophysiological experiments on the cell body of the cockroach (Periplaneta americana) fast coxal depressor motor neuron (Df), and GABA-activated 36Cl- uptake experiments on microsacs prepared from cockroach ventral nerve cords showed that both heptachlor and heptachlor epoxide blocked functional GABA receptors. The block appeared to be non-competitive and was voltage-independent over the membrane potential range -75 mV to -110 mV. There was no significant difference between the potencies of heptachlor and heptachlor epoxide in the functional assays for insect GABA receptors. Both compounds inhibited [35S]-t-butylbicyclophosphorothionate [( 35S]TBPS) binding in insects and vertebrates. The findings provide further evidence for block of an insect GABA receptor/Cl- channel by the cyclodiene class of polychlorocycloalkanes, and reveal differences in the insecticide-[35S]TBPS binding site interactions of insects and vertebrates.

Cell proliferation in the repairing adult insect central nervous system: incorporation of the thymidine analogue 5-bromo-2-deoxyuridine in vivo

Uptake of the thymidine analogue 5-bromo-2-deoxyuridine into non-neuronal cells of the insect central nervous system has been examined following a controlled lesioning of the glial elements. The pattern of BUdR labelling along the penultimate abdominal connective was examined over a period of 17 days. Cell proliferation occurred in and immediately around the site of damage in both perineurial and subperineurial glial cells but at different times post-lesion for the two regions. Proliferation in the perineurial zone was maximal at 6-8 days post-lesion but continued for at least 17 days. Subperineurial proliferation was less dramatic and peaked between days 8-11 post-lesion. In both areas division appears to be confined to the reactive glial cells. These results are discussed in the context of past research on this system, particularly with regard to the restoration of the blood-brain barrier.