Ionic events following GABA receptor activation in an identified insect motor neuron

The ionic events underlying gamma-aminobutyric acid (GABA) receptor activation on the cell body of a cockroach identified motor neuron were investigated by using current-clamp and voltage-clamp techniques. The reversal potential for GABA-induced hyperpolarization was -77.0 +/- 2.4 mV (mean +/- s.e.m.; n = 22). The reversal potential for GABA was highly sensitive to changes in external chloride, only weakly affected by changes in external potassium, and independent of changes in either sodium or calcium ion concentration. Intracellular ion-sensitive microelectrodes confirmed that an influx of chloride ions mediated the GABA response. Intracellular injection of acetate, citrate, sulphate, fluoride or ammonium caused no change in the reversal potential for GABA. Intracellular injection of chloride, bromide, chlorate, bromate, or methyl sulphate shifted the reversal potential for GABA to values more positive than resting membrane potential. Evidence for chloride accumulating and for extrusion mechanisms was examined by using putative inhibitors. However, internal application of ammonium ions, and external application of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), acetazolamide, furosemide, ammonium, zinc and copper ions, were all without effect on the reversal potential for GABA.     

Pharmacology of insect GABA receptors

A GABA-operated Cl^– channel that is bicuculline-insensitive is abundant in the nervous tissue of cockroach, in housefly head preparations and thorax/abdomen preparations, and in similar preparations from several insect species. Bicuculline-insensitive GABA-operated Cl^– channels, which are rare in vertebrates, possess sites of action of benzodiazepines, steroids and insecticides that are pharmacologically-distinct from corresponding sites on vertebrate GABA_A receptors. The pharmacological profile of the benzodiazepine-binding site linked to an insect CNS GABA-operated Cl^– channel resembles more closely that of vertebrate peripheral benzodiazepine-binding sites. Six pregnane steroids and certain polychlorocycloalkane insecticides, which are active att-butylbicy-clophosphorothionate (TBPS)-binding sites, also differ in their effectiveness on vertebrate and insect GABA receptors. Radioligand binding and physiological studies indicate that in insects there may be subtypes of the GABA receptor. Molecular biology offers experimental approaches to understanding the basis of this diversity.Special issue dedicated to Dr. Eugene Roberts     

Competitive antagonism of insect GABA receptors by iminopyridazine derivatives of GABA

A series of 4-(6-imino-3-aryl/heteroarylpyridazin-1-yl)butanoic acids were synthesized and examined for antagonism of GABA receptors from three insect species. When tested against small brown planthopper GABA receptors, the 3,4-methylenedioxyphenyl and the 2-naphthyl analogues showed complete inhibition of GABA-induced fluorescence changes at 100 μM in assays using a membrane potential probe. Against common cutworm GABA receptors, these analogues displayed approximately 86% and complete inhibition of GABA-induced fluorescence changes at 100 μM, respectively. The 4-biphenyl and 4-phenoxyphenyl analogues showed moderate inhibition at 10 μM in these receptors, although the inhibition at 100 μM was not complete. Against American cockroach GABA receptors, the 4-biphenyl analogue exhibited the greatest inhibition (approximately 92%) of GABA-induced currents, when tested at 500 μM using a patch-clamp technique. The second most active analogue was the 2-naphthyl analogue with approximately 85% inhibition. The 3-thienyl analogue demonstrated competitive inhibition of cockroach GABA receptors. Homology modeling and ligand docking studies predicted that hydrophobic 3-substituents could interact with an accessory binding site at the orthosteric binding site.     

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.     

Neuromodulation of vertebrate motor neuron membrane properties

The short-term function of motor neurons is to integrate synaptic inputs converging onto the somato-dendritic membrane and to transform the net synaptic drive into spike trains. A set of voltage-gated ion channels determines the electro-responsiveness and thereby the motor neuron's input-output function. In addition, several of the decisive ion channels are transmitter controlled, which results in a flexible control of the input-output relationship.     

Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone

The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D_f) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D_f motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.     

Changes in functional glutamate receptors on a postsynaptic neuron accompany formation and maturation of an identified synapse

N-Methyl-D-aspartate (NMDA)-type glutamate receptors play important roles at developing synapses and in activity-dependent synaptic plasticity. Recent studies in Aplysia suggest that NMDA-like receptors may contribute to some forms of plasticity of sensorimotor synapses accompanying associative learning. We examined at various times after plating neurons in culture the contribution of NMDA- and alpha-amino-3 hydroxy-5 methyl-4 isoxazole proprionic acid (AMPA)-like glutamate receptors to responses evoked in motor cell L7 either by action potentials in sensory neurons (SNs) or by focal applications of glutamate. We found that (D,L)-2-amino-5-phosphopentoic acid-sensitive receptors contributed significantly to postsynaptic responses in 1-day cultures but contributed little in the same cultures on day 4. By contrast, postsynaptic responses on day 4 increased significantly in amplitude by the addition of functional 6-cyano-7 nitroquinoxaline-2,3-dione- or 1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine hydrochloride-sensitive receptors. Receptors with NMDA-like properties are detected on day 1 only at sites on L7 apposed to SN varicosities, and are not detected on L7 cultured alone. The results indicate that changes in expression and distribution of functional receptors on L7 accompany the formation and maturation of SN synapses. Signals from the SN appear to trigger expression and clustering of functional NMDA-like receptors at sites contacted by presynaptic structures capable of transmitter release. With time, functional AMPA-like receptors are added to these sites enhancing synaptic efficacy. The results are consistent with the idea that the expression and sequential clustering of NMDA- and AMPA-type receptors may be essential for the formation and maturation of central synapses.     

An axonal spike in an identified fast crab motor neuron has sodium and calcium components

1. High intensity intracellular stimulation of the FBE axon produced a spike discharge. The spikes divided into two components, each with a different frequency. 2. Ion replacement and blocking specific ion channels revealed that the FBE spike has calcium and sodium components. 3. The pronounced depolarizing wave that follows the FBE spike is not produced by changes in calcium conductance.     

Effect of fenibut on the GABA B receptors of the spinal motor neurons

It has been established in experiments on the isolated spinal cord of 7-14-day-old rats that the GABAB-mimetic phenibut (10(-5)--10(-4) M) elicits a slow-developing depolarization of motoneurons, suppression of spontaneous activity and polysynaptic reflex discharges of motoneurons, recorded from the ventral roots. Administered under the same conditions GABA produces de- and hyperpolarization of motoneurons. The depolarization of motoneurons elicited by phenibut and GABA is not reversed by picrotoxin in contradistinction to the GABA-induced hyperpolarization of motoneurons, being associated with a direct action of the GABA-mimetics on postsynaptic GABAB receptors of motoneurons. Diazepam (10(-9)--10(-6) M) potentiates the effects of phenibut supposedly via benzodiazepine receptors bound with GABAA receptors (an independent interaction).     

The timing of initial neuropeptide expression by an identified insect neuron does not depend on interactions with its normal peripheral target

To study the developmental regulation of a neuropeptide phenotype, we have analyzed the biochemical and morphological differentiation of two identifiable neurons in embryos of the moth, Manduca sexta. The central cell, CF, and the peripheral cell, L1, are both neuroendocrine neurons that express neuropeptides related to the molluscan tetrapeptide FMRFamide. Both neurons project axons to the transvers nerve in each thoracic segment. Within the CF and L1 cells, neuropeptide-like immunoreactivity was localized to secretory granules that had cell specific morphologies and sizes. The onset of neuropeptide expression in the two cell types displayed a similar pattern: immunoreactivity was first detected in distal processes and soon after within cells bodies. However, the onsets occurred at different times: for the CF cell, neuropeptides were first seen at 60%-63% of embryonic development, after the neuron had extended a long axon into the periphery, while L1 neuropeptide expression began at ～42%, as it first extended its growth cone. These times were related in that they corresponded to the arrival times of the respective growth cones at a similar position in the developing peripheral nerve. Withinthis region of the nerve, the growth cones of both cell typesexhibited a transient and cell-specific interaction with an identified mesodermal cell, called the Syncytium. Like the L1 and B neurons (Carr and Taghert, 1988b), the CF growth cones typically grew past this cell, yet remained attached to it by lamellipodial and filopodial processes of the axon. Ultrastructurally, the interaction involved filopodial adhesion to and insertion within the Syncytial cell. Two other nonneuroendocrine cell types grew axons past this same region, but showed no such tendencies. To test the hypothesis that the morphological and biochemical differentiation of these cells was somehow linked, central ganglia were isolated (as individuals or connected as ganglionic chains) in tissue culture, prior to the time when CF growth cones entered the periphery and prior to the development of CF neuropeptide expression. In the majority of cases, CF neurons nevertheless displayed their neuropeptide phenotype at a normal and cell-specific stage. We conclude that the initiation of neuropeptide expression is highly correlated with schedules of morphological differentiation in these neurons, but that, in the case of the CF neuron, it is not regulated by interactions of the growth cone with peripheral structures.     

Characterization of the interaction between fenamates and hippocampal neuron GABA(A) receptors

Fenamate NSAIDs have several central effects, including anti-epileptic and neuroprotective actions. The underlying mechanism(s) of these actions are not presently understood. In this study, the effects of five members of the fenamate NSAID group were investigated on native ligand-gated ion channels expressed in cultured rat hippocampal neurons. All fenamates tested (1-100 microM) dose-dependently potentiated GABA-evoked currents; mefenamic acid (MFA) was the most potent and efficacious and was found to shift the GABA dose-response curve to the left without effect on the maximum amplitude or the GABA Hill Slope. The modulation of GABA receptors by MFA was not reduced in the presence of the benzodiazepine antagonist, flumazenil (10 microM) and was moderately voltage-dependent. MFA at concentrations >or=10 microM evoked dose-dependent currents in the absence of GABA. These currents were potentiated by diazepam (1 microM) and blocked by bicuculline (10 microM). The MFA (50 microM) current-voltage relationship and reversal potential were similar to that evoked by GABA. MFA (1-100 microM) had no effects on sub-maximal glycine, glutamate or NMDA evoked currents. These data show that fenamate NSAIDs are a highly effective class of GABA(A) receptor modulator and activators.     

The timing of initial neuropeptide expression by an identified insect neuron does not depend on interactions with its normal peripheral target.

To study the developmental regulation of a neuropeptide phenotype, we have analyzed the biochemical and morphological differentiation of two identifiable neurons in embryos of the moth, Manduca sexta. The central cell, CF, and the peripheral cell, L1, are both neuroendocrine neurons that express neuropeptides related to the molluscan tetrapeptide FMRFamide. Both neurons project axons to the transverse nerve in each thoracic segment. Within the CF and L1 cells, neuropeptide-like immunoreactivity was localized to secretory granules that had cell-specific morphologies and sizes. The onset of neuropeptide expression in the two cell types displayed a similar pattern: immunoreactivity was first detected in distal processes and soon after within cell bodies. However, the onsets occurred at different times: for the CF cell, neuropeptides were first seen at 60%-63% of embryonic development, after the neuron had extended a long axon into the periphery, while L1 neuropeptide expression began at approximately 42%, as it first extended its growth cone. These times were related in that they corresponded to the arrival times of the respective growth cones at a similar position in the developing peripheral nerve. Within this region of the nerve, the growth cones of both cell types-exhibited a transient and cell-specific interaction with an identified mesodermal cell, called the Syncytium. Like the L1 and B neurons (Carr and Taghert, 1988b), the CF growth cones typically grew past this cell, yet remained attached to it by lamellipodial and filopodial processes of the axon. Ultrastructurally, the interaction involved filopodial adhesion to and insertion within the Syncytial cell. Two other nonneuroendocrine cell types grew axons past this same region, but showed no such tendencies. To test the hypothesis that the morphological and biochemical differentiation of these cells was somehow linked, central ganglia were isolated (as individuals or connected as ganglionic chains) in tissue culture, prior to the time when CF growth cones entered the periphery and prior to the development of CF neuropeptide expression. In the majority of cases, CF neurons nevertheless displayed their neuropeptide phenotype at a normal and cell-specific stage. We conclude that the initiation of neuropeptide expression is highly correlated with schedules of morphological differentiation in these neurons, but that, in the case of the CF neuron, it is not regulated by interactions of the growth cone with peripheral structures.     

Dynamics and metamorphosis of an identifiable peptidergic neuron in an insect

Eclosion hormone (EH) is a 7000 Da peptide that triggers ecdysis behavior in insects. In the moth, Manduca sexta, EH is found in two pairs of ventromedial (VM) cells in the brain which send their axons down the ventral nerve cord to a neurohemal site in the proctodeal nerve in the larva and pupa. During adult development, these cells send axon collaterals to the corpora cardiaca where they form a new release site used for adult eclosion. Studies of bioassayable peptide during the 5th larval instar and the larval-pupal transformation revealed that after depletion at ecdysis, the VM cells showed a transient increase in EH found in their cell bodies and axons. By contrast, their terminals in the proctodeal nerve showed a gradual accumulation of peptide followed by a release of over 90% of the stored material at pupal ecdysis. In situ hybridization analysis on whole mounts of the brains showed that the VM cells always contained EH mRNA with increased accumulation during the larval and pupal molting periods with a slight decline just before ecdysis. High levels of EH mRNA were found in brains of diapausing pupae. During the first two-thirds of adult development, mRNA accumulated to high levels, then slowly declined until ecdysis. EH mRNA levels up to 3 days after adult eclosion. At no time was EH mRNA found in the lateral neurosecretory cell cluster previously reported to produce EH for adult eclosion. 1994 John Wiley & Sons, Inc.     

Structural organization of an identified giant neuron of Helix lucorum

Method of intracellular staining with cobalt was used for detailed study of processes branching of the giant cell in the left parietal ganglion of the snail Helix lucorum L. Dendritic and axonal branches are described and quantitatively characterized. Terminals of axonal collaterals of this neurone innervating presumed neurosecretory bodies are described in the tissue surrounding the ganglion.     

Effects of pharmacological treatment and photoinactivation on the directional responses of an insect neuron

Soma-ipsilateral branches of the large segmental omega neuron of the phaneropterid bush cricket Ancistrura nigrovittata have smooth endings, which extend through most of the auditory neuropile. Correspondingly, it shows a broad frequency tuning. Large excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) are observed when recording from soma-ipsilateral branches. Stimulation from the soma-ipsilateral side leads to a strong excitation. Soma-contralateral branches have a strong, beaded appearance. IPSPs, which seem to be of soma-contralateral origin, can be recorded from these branches. Stimulation from the soma-contralateral side leads to a strong inhibition of the omega neuron. Soma-contralateral stimulation must be 30-40 dB more intense than soma-ipsilateral stimulation to evoke similar spike numbers in the omega neuron. The side-to-side difference is reduced to 10-15 dB after cutting the input from the soma-contralateral leg (tympanic nerve). The thresholds for eliciting IPSPs by soma-contralateral stimulation correspond roughly to excitatory thresholds of the mirror-image omega with the same stimuli. Pharmacological treatment with picrotoxin (PTX) or photoinactivation of the Lucifer Yellow filled mirror-image omega neuron reduces contralateral inhibition considerably and eliminates all visible IPSPs. Nevertheless, an additional contralateral inhibition survives both procedures and is only eliminated after cutting the soma-contralateral tympanic nerve. These results demonstrate that the mirror-image partners of the omega neuron mutually inhibit each other in bush crickets--as in crickets. This mutual inhibition is PTX-sensitive. At least one additional element exerts contralateral PTX-insensitive inhibition on the omega neuron.     

Ouabain-sensitive and insensitive acetylcholine receptors on the membrane of the same neuron in Helix pomatia

Using internally dialyzed neurons of Helix pomatia as a model, the effect of structural analogs of acetylcholine (ACh) were investigated for their cholinomimetic properties on A- and B-types of ACh-responses. Specifically we analyzed choline esters of N-para- and ortho-alkoxybenzoyl-beta-alanines, (CH(3)O-, C(2)H(5)O-, C(3)H(7)O-, iso-C(3)H(7)O-, C(4)H(9)O-, iso-C(4)H(9)O-, C(5)H(11)O-, iso-C(5)H(11)O-), (in all, 16 combinations). The compounds evoked differing sensitivities of response to factors de-activating the Na-K-pump (ouabain and K-free solution). Most compounds resembled ACh: ionic currents caused by these compounds were inhibited by Na-K pump blockers in the case of A-type responses - B-type responses were insensitive to these factors. Ionic currents induced by choline esters of p-, o-propoxy- and iso-propoxybenzoyl-beta-alanines were insensitive to ouabain and K-free solution in the case of A- and B-type responses. Ionic currents induced by the choline ester of p-butoxybenzoyl-beta-alanine were inhibited by ouabain and K-free solution on both types of neuron. The results allow us to classify choline esters of p-, o-propoxy- and iso-propoxybenzoyl-beta-alanines as N-cholinomimetics, while the choline ester of p-butoxybenzoyl-beta-alanine can be considered as M-cholinomimetic. We conclude that both M- and N-type ACh receptors exist on the membrane of the same neurons.