Insect neuronal cultures: an experimental vehicle for studies of physiology, pharmacology and cell interactions.

The current status of insect neuronal cultures is discussed and their contribution to our understanding of the insect nervous system is explored. Neuronal cultures have been developed from a wide range of insect species and from all developmental stages. These have been used to study the morphological development of insect neurones and some of the extrinsic factors that affect this process. In addition, they have been used to investigate the physiology of sodium, potassium and calcium channels and the pharmacology of acetylcholine and GABA receptors. Insect neurones have also been grown in culture with muscle and glial cells to study cell interactions.

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 method, 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.

Synthesis of functional GABAA receptors in stable insect cell lines.

We have synthesised the beta 1-subunit of the bovine GABAA receptor in stable, continuous insect (Spodoptera frugiperda) cell lines. A cDNA was integrated randomly into the insect cell genome under control of a baculovirus immediate early (IE-1) gene promoter. Transformed cells were obtained by co-transfection of the insect cells with pIEK1.GR beta 1, encoding the beta 1 subunit cDNA, and pIEK1.neo, encoding the neomycin resistance gene. G-418-resistant clones were selected and expanded into continuous cell lines synthesising functional, GABA-gated, homo-oligomeric chloride channels. These cell lines had significant advantages over the transient baculovirus expression system for the characterisation of receptors using electrophysiological recording techniques.

Mutational analysis of the mouse 5-HT7 receptor: importance of the third intracellular loop for receptor-G-protein interaction.

The mouse serotonin (5-HT) receptor subtype, 5-HT7, belongs to the family of seven transmembrane G-protein-coupled receptors. To identify the structural basis for the coupling of 5-HT7 receptor to G alpha(s) we constructed a number of receptor mutants in which amino acid residues were either substituted or deleted from the second and third intracellular loops. Wild-type and mutant 5-HT7 receptors were expressed in insect cells using the baculovirus vectors. Two mutant receptor species, 5-HT7(E325G) and 5-HT7(K327S), demonstrated markedly impaired abilities to stimulate adenylyl cyclase. The results suggest the importance of the C-terminal region of the third intracellular loop in receptor-G-protein interaction and that specific charged residues, E325 and K327, may play a critical role in this interaction.

Functional characterisation of the Drosophila 5-HTdro1 and 5-HTdro2B serotonin receptors in insect cells: activation of a G(alpha) s-like protein by 5-HTdro1 but lack of coupling to inhibitory G-proteins by 5-HTdro2B.

Insect cells are routinely used for the production of receptor proteins. Expression of the Drosophila 5-HTdro1 serotonin receptor resulted in positive coupling of the receptor to adenylyl cyclase via the G(alpha)s G-protein subtype. The Drosophila 5-HTdro2B receptor stimulated the metabolism of inositol phospholipid via a pertussis toxin-insensitive G-protein, but exhibited no detectable inhibition of adenylyl cyclase. Immunoblot analysis of the endogenous G-proteins revealed that Sf9 cells lack the G-protein subtypes G(alpha i 1-3) and G(alpha)o, but express the subtype G(alpha)s and G(alpha)q.

Effects of diltiazem on human nicotinic acetylcholine and GABA(A) receptors.

Effects of the L-type calcium channel antagonist diltiazem on recombinant human GABA(A) receptor (alpha1beta2gamma2s) or on muscle (alpha1beta1deltagamma and alpha1beta1delta(epsilon)) or neuronal (alpha7 and alpha4beta2) nicotinic acetylcholine receptors expressed in Xenopus oocytes were examined using two-electrode voltage-clamp. Diltiazem inhibited the function of both muscle and neuronal nicotinic receptors, but it had no effect on GABA(A) receptors. The extent of functional inhibition of nicotinic receptors depended on the receptor subtype, and the order of inhibition potency by diltiazem was alpha7>alpha4beta2 approximately alpha1beta1deltagamma approximately alpha1beta1delta(epsilon). Inhibition of alpha7 receptor function was non-competitive and voltage-independent, and it occurred at concentrations far lower than those needed to inhibit (never completely) binding of (125)I-alpha-bungarotoxin to heterologously expressed alpha7 receptors in mammalian cells. Pre-incubation in diltiazem before concomitant application with acetylcholine increased inhibition of function and slowed recovery from inhibition. Verapamil, a phenylalkylamine antagonist of L-type Ca(2+) channels also fully inhibited alpha7 receptor function and partially inhibited (125)I-alpha-bungarotoxin binding to alpha7 receptors, but was less potent than diltiazem. Effects on both alpha7 receptor function and (125)I-alpha-bungarotoxin binding by verapamil plus diltiazem suggest separate sites for verapamil and diltiazem on alpha7 receptors. These results provide further evidence that L-type Ca(2+) channel drugs inhibit ligand-gated cationic channels and suggest that caution should be applied when using these compounds to study systems in which L-type Ca(2+) channels and ligand-gated cationic channels co-exist.

Assembly of functional GABAA receptors in insect cells using baculovirus expression vectors.

We have constructed recombinant baculoviruses containing cDNAs encoding either the alpha 1- or the beta 1-subunit of the bovine GABAA receptor. In Spodoptera frugiperda (IPLB-Sf-21) cells infected with recombinant virus expressing either the alpha 1- or beta 1-subunit, or in cells co-infected with both viruses, functional GABAA receptors were detected by whole-cell electrophysiological recordings. The threshold for the responses mediated by the homo-oligomeric channels (alpha- or beta-) was 2-3 x 10(-6) M GABA, and for the co-infected cells was 8 x 10(-8) M GABA, suggesting that hetero-oligomeric channels formed in these cells. All GABA-induced currents were found to be inhibited by bicuculline and picrotoxin, potentiated by pentobarbital but were insensitive to benzodiazepines.

Dihydroavermectin B1: actions on cultured neurones from the insect central nervous system.

The physiological effects of dihydroavermectin B1 on insect central neurones have been investigated using a culture system derived from the brains of embryonic cockroaches. In these neuronal cultures 60% of the cells respond to the application of gamma-aminobutyric acid (GABA) with a conductance increase; these responses are blocked by picrotoxin but not by bicuculline. Dihydroavermectin B1, a representative of a potentially new class of insecticide, also produces a slow conductance increase which is blocked by picrotoxin and inverted by the injection of chloride ions. Qualitatively similar responses are also evoked by dihydroavermectin B1 in some neurones unaffected by GABA and in neurones exposed to elevated Mg2+ concentrations to inhibit synaptic release mechanisms. In a subpopulation of neurones dihydroavermectin B1 evokes a transient, initial excitation prior to the apparent chloride conductance increase.

Cholinergic receptors on cultured neurones from the central nervous system of embryonic cockroaches.

Cultured neurones from the cockroach, Periplaneta americana, have been used to investigate putative acetylcholine receptors. Ligand-binding experiments revealed that these neurones possessed an alpha-bungarotoxin binding site that was saturable, had an apparent affinity constant of 3.51 nM and was predominantly nicotinic in nature. An individual culture of 50,000 neurones had a maximum of 4200 pmol. binding sites per gram of protein. [I125]alpha-BTX autoradiography showed the binding sites to be distributed over both the neuronal cell bodies and their associated axonal processes. Both acetylcholine and nicotine applied by pressure ejection to the neuronal soma induced depolarizing responses and in the majority of cells tested the response was blocked by alpha-BTX at a concentration of 25 nM in a time dependent manner. Some of the neurones, however, were depolarized by acetylcholine and nicotine after 3 h incubation in alpha-BTX. These experiments suggest that two populations of cells possessing extrajunctional nicotinic receptors were present in these cultures. In the majority of cells these receptors were sensitive to alpha-BTX but in a subpopulation the receptors were unaffected by this toxin.

Responses to GABA by isolated insect neuronal somata: pharmacology and modulation by a benzodiazepine and a barbiturate.

Mechanically dissociated neuronal somata from the thoracic ganglia of Locusta migratoria and Schistocerca gregaria were viable in vitro for hours and were current- and voltage-clamped to record the responses evoked by brief pressure applications of gamma-aminobutyric acid (GABA) in the presence of various modulators. The application of GABA and muscimol, but not baclofen, produced a hyperpolarization and concurrent increase in the membrane conductance. The current underlying this response reversed at -65 mV, was evoked in all cells tested and showed outward rectification. In 6 of 74 Locusta neurones but not in the neurones of Schistocerca, GABA and muscimol evoked a biphasic response. The initial, fast phase was indistinguishable from the GABA-evoked current seen in all neurones. The remaining predominant, slow and long-duration component of the response was an inward current over the membrane potential range 0 to -80 mV, increasing with hyperpolarization. The GABAA antagonists bicuculline and pitrazepin were without effect on the fast GABA response while picrotoxin was a potent blocker of both the fast and the slow GABA responses. Flunitrazepam enhanced the amplitude of the fast response by up to 70% without increasing its duration. Sodium pentobarbital enhanced both the amplitude and the duration of the fast GABA response. We conclude that the locust thoracic neuronal GABA receptor/channel complex resembles the vertebrate GABAA receptor in having associated modulatory receptor sites for benzodiazepines and barbiturates, but differs from it in terms of the pharmacology of the GABA receptor itself.

The effects of L-glutamate on cultured insect neurones.

The effects of L-glutamate on insect cultured neurones were studied under current and voltage-clamp conditions using conventional and whole-cell patch-clamp techniques. Brief pressure or iontophoretic application of L-glutamate produced either a depolarisation or hyperpolarisation. The current underlying the depolarisation was inwardly directed and reversed at around 0 mV while the hyperpolarisation was caused by an outward current that reversed between -60 and -80 mV. Single channel currents underlying the depolarisation were readily recorded from cell attached patches and showed multiple conductance states. Channel activity corresponding to the hyperpolarising response has not yet been observed.

Voltage-dependent calcium channel subtypes controlling somatic substance P release in the peripheral nervous system.

1. Isolated rat dorsal root ganglia (DRG) neurones support vesicular, non synaptic release of substance P in a depolarisation and Ca2+ dependent manner. 2. In vivo this process may mediate cross-communication between DRG cells in some neuropathological conditions and is therefore a putative area for drug intervention. 3. The authors investigated the voltage-dependent Ca2+ channel (VDCC) subtypes involved in somatic release of substance P. Fresh (< 1 day) cultures of DRG neurones were incubated with high K+ depolarising saline in the presence and absence of subtype selective VDCC blockers. Substance P released into the external media was collected and quantified using a radioimmunoassay. 4. The results show that L-type and N-type, but not P-type, VDCCs play an important role in high K+ evoked substance P release from rat DRG neurones.

The morphology and ultrastructure of antennal lobe cells from pupal honeybees (Apis mellifera) growing in culture.

A culture technique for the in vitro growth and differentiation of antennal lobe cells from the honeybee, Apis mellifera, is described and the ultrastructure of the growing cells is analysed. Two types of cell are present in the cultures and from their morphology and ultrastructure they can be identified as glial cells and neurones. The neurones have a granular cytoplasm, abundant endoplasmic reticulum and a small, densely stained nucleus. They produce long processes with varicosities that contain dense-core and clear vesicles. In contrast the glial cells have clear cytoplasm, little endoplasmic reticulum and a distinct cytoskeletal organisation. These cells produce short, flat processes that spread over the surface of the culture dish. Although a number of cell contacts have been identified in the cultures no synapses have yet been seen. These cultures provide a good in vitro model for an analysis of the interactions between cells derived from the antennal lobe of the honey bee.

Cockroach glial cell cultures: morphological development and voltage-gated potassium channels.

Insect glial cells derived from the embryonic brain of Periplaneta americana cockroaches have been kept in primary culture conditions for up to 3 weeks. Under the culture conditions used, the glial cells differentiated and formed a complex cellular network on the floor of the culture vessels from which glial-glial and glial-neuronal contacts could be seen. Single-channel currents from cell-attached glial membrane patches were recorded using the gigaseal technique. Depolarisation of membrane patches activated outward currents, which were abolished in the presence of extracellular 50 mM tetraethylammonium or 5 mM 4-aminopyridine, and were insensitive to 1 microM tetrodotoxin, 10 microM picrotoxin and 2 mM Cd2+. The amplitude of the outward currents increased linearly with depolarisation, and amplitude histograms obtained at several pipette potentials could be reasonably fitted with a single Gaussian corresponding to a single channel type with a slope conductance of 37 +/- 11 pS. The extrapolated equilibrium potential of the outward current was about 5 +/- 10 mV positive to the resting potential and both the channel open time constant and relative open time probability were sensitive to membrane potential, increasing markedly with depolarisation. The results presented in this paper show the presence of a cadmium-insensitive, voltage-dependent outward potassium channel in insect glial cells in vitro.

Neurite regeneration of long-term cultured adult insect neurosecretory cells identified as DUM neurons.

A distinctive group of neurons having cell bodies located along the midline of the dorsal surface of the sixth abdominal (A6) ganglion of the adult cockroach Periplaneta americana has been characterized by direct anterogradc cobalt chloride staining. These neurons identified as dorsal unpaired median (DUM) neurons, present a T-shaped morphology. The soma gives rise to a single primary neurite running anteriorly in the ganglion before dividing into two lateral neurites which run into the left and the right side of the ganglion. A characteristic dendritic arborization arises from the lateral neurites within the ganglion. This major branching pattern is mainly located at the periphery of the A6 ganglion and forms a symmetrical complicated network. A new culture procedure of these same adult DUM neurons has been developed from the dissociation of the median parts of the A6 ganglia. In our experimental conditions, we show that cultured adult DUM neurons can survive for several weeks, and regenerate a single primary neurite dividing into two symmetrical lateral neurites with a number of fine processes radiating from the endings. This corresponds to the typical DUM neuron morphology revealed in situ on the same preparation using the cobalt chloride staining technique. This culture system developed for the first time on A6 ganglia adult DUM neurons will allow a better understanding of the physiological intracellular mechanisms involved in the neurosecretory functions of DUM neurons, which are currently unknown.

Morphological analysis of honeybee antennal cells growing in primary cultures.

A culture technique for the in vitro growth of antennal cells from honeybee is described. On the basis of morphological and immunocytochemical criteria, the cultured cells could be classified into neural and non-neural cells. Neural cells (type D) exhibited the main morphological features of insect olfactory receptor neurones (ORNs). Non-neural cells were large, flat cells that could be divided into three main types: Type A, B and C cells. Type A cells were spindle-like cells and resembled insect myocytes in culture. Type B cells were large cells with a veil-like cytoplasm. These cells tended to group and vacuolate towards the center of the cellular aggregate. Type C cells were either bipolar (Type C1) or multipolar (Type C2) flat cells which closely resembled insect glial cells in cultures.

Ultrastructure of the accessory glands of gene's organ in the cattle tick, Boophilus.

The organization and ultrastructure of the accessory glands of the cattle tick, Boophilus microplus, are described. The glands consist of two groups of acinar cells situated on either side of Gene's organ. A single acinus consists of from eight to 12 cells and each cell is connected via an individual duct to pores on the dorsal surface of the mouthparts. The position of these pores is such that the secretion of the accessory glands is incorporated into the egg wax during oviposition. Each gland cell has striking quantities of smooth endoplasmic reticulum and numerous Golgi dictyosomes and appears to produce a secretion that is lipoidal in nature. Each cell secretes into its own individual lumen and is connected to a cuticular pore by a duct cell.

Intercellular junctions in the hypodermis, salivary gland and Gené's organ of the cattle tick, Boophilus microplus.

The intercellular junctions that occur in the hypodermis, Gené's organ, and the salivary glands of the tick, B. microplus, are described. The epithelial cells of the hypodermis are connected by spot desmosomes and septate junctions and the secretory cells of Gené's organ by septate and gap junctions. The cap cells in the alveoli of the salivary gland connect to adjacent cells by gap junctions, hemidesmosomes and septate junctions into which microtubules are inserted.

Morphology of identified cercal afferents and giant interneurones in the hatchling cockroach Periplaneta americana.

Cobalt backfills from the thoracic connectives of the hatchling Periplaneta americana allowed identification of giant interneurones in the terminal abdominal ganglion, morphologically comparable to GI 1, 2 and 3 in the adult. The bipolar neurone innervating each cercal filiform wind receptor hair is ultrastructurally similar to the adult cell and possesses an individually identifiable afferent axon, four of which provide the behaviourally functional escape response system with a simplified sensory input. Both pre- and postsynaptic neurones can be identified and may provide a good preparation for the study of cholinergic synapses.

The pharmacological profile of GABA receptors on cultured insect neurones.

Neuronal cultures of the cockroach, Periplaneta americana, were used to study the pharmacological profile of GABA receptors using the whole-cell-voltage clamp technique. The results indicated that insect GABA receptors are linked to a chloride channel that can be activated by both GABA(A) and GABA(C) receptor agonists. The receptors are blocked by GABA(A) chloride channel blockers and some insecticides but not by competitive GABA(A) receptor antagonists. The GABA(C) receptor competitive antagonists were either full or partial agonists of the cockroach GABA receptors. The receptors were modulated by the enantiomers of lindane. In conclusion, insect GABA receptors appear to have a distinct pharmacological profile that does not conform to either vertebrate GABA(A) or GABA(C) receptors.