Molecular cloning of a GABA receptor subunit from Laodelphax striatella (Fallén) and patch clamp analysis of the homo-oligomeric receptors expressed in a Drosophila cell line.

A cDNA encoding a gamma-aminobutyric acid (GABA) receptor subunit was cloned from the small brown planthopper Laodelphax striatella. The L. striatella GABA receptor subunit was found to have high amino acid sequence similarity to the bd-type splice variant of the Drosophila GABA receptor Rdl subunit and several other GABA receptor subunits, with identities of over 70%. The cDNA was inserted into the expression vector pAc5.1-lac-Hygro. Clonal cell lines stably expressing homo-oligomeric L. striatella GABA receptors were generated by transfecting the vector into D.mel-2 cells. Expression of functional GABA receptors in the cell lines was demonstrated by whole-cell patch clamp recordings. GABA induced inward currents with an EC(50) value of 29 microM and a Hill coefficient of 1.7. The GABA-evoked responses reversed close to the Nernst equilibrium potential for chloride ions. The amplitudes of agonist-induced currents were found to be in the order muscimol (100 microM) >/= GABA (100 microM) > isoguvacine (100 microM) > cis-4-aminocrotonic acid (CACA) (100 microM) > 5-(4-piperidyl)-3-isoxazolol (4-PIOL) (1 mM). Antagonists such as fipronil (100 nM), 4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) (100 nM), dieldrin (100 nM) and SR95531 (gabazine) (1 microM) suppressed GABA-induced currents. The functional expression of a GABA receptor from an agricultural pest presents a unique opportunity to discover new molecules active at this important target site.

Molecular cloning and heterologous expression of an alpha-adrenergic-like octopamine receptor from the silkworm Bombyx mori.

A cDNA encoding an octopamine (OA) receptor (BmOAR1) was isolated from the nerve tissue of silkworm (Bombyx mori) larvae. Comparison of amino acid sequences showed that BmOAR1 is highly identical to OA receptors isolated from Periplaneta americana (Pa oa(1)), Apis mellifera (AmOA1), and Drosophila melanogaster (OAMB or DmOA1A). BmOAR1 was stably expressed in HEK-293 cells. OA above 1 microM led to an increase in intracellular cyclic AMP concentration ([cAMP](i)). The synthetic OA-receptor agonist demethylchlordimeform also elevated [cAMP](i) to the same maximal level (approximately 5-fold over the basal level) as that induced by OA. However, other biogenic amines, tyramine and dopamine, and chlordimeform were without effects. The [cAMP](i) level raised by OA was lowered by antagonists; the rank order of antagonist activity was chlorpromazine > mianserin = yohimbine. Cyproheptadine and metoclopramide had little effect. OA above 100 nM induced a transient or sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), depending on the concentration of OA. Sequence homology and functional analysis data indicate that BmOAR1 is an alpha-adrenergic-like OA receptor of B. mori.

Linalool suppresses voltage-gated currents in sensory neurons and cerebellar Purkinje cells.

Linalool is a major component of essential oils and possesses various biological effects in sensory or central nervous systems. To investigate the pharmacological and biophysical effects of linalool on voltage-gated currents in sensory neurons, we used the whole-cell patch clamp and the Ca(2+) imaging techniques. Under the voltage clamp, membrane depolarization generated time- and voltage-dependent current responses in newt olfactory receptor cells (ORCs). Linalool significantly and reversibly suppressed the voltage-gated currents in ORCs. The dose-suppression relation of linalool for the voltage-gated Na(+) current could be fitted by the Hill equation with a half-blocking concentration of 0.56 mM and a Hill coefficient of 1.2. To test whether linalool suppresses voltage-gated currents in ORCs specifically or suppresses currents in other neurons generally, we next examined the effects of linalool on voltage-gated currents in newt retinal neurons and rat cerebellar Purkinje cells. Linalool suppressed the voltage-gated currents not only in retinal horizontal cells and ganglion cells but also in Purkinje cells. Furthermore, bath application of linalool inhibited the KCl-induced [Ca(2+)](i) response of ORCs, suggesting that linalool suppresses Ca(2+) currents in ORCs. These results suggest that linalool non-selectively suppresses the voltage-gated currents in newt sensory neurons and rat cerebellar Purkinje cells.

Synaptic modulation contributes to firing pattern generation in jaw motor neurons during rejection of seaweed in Aplysia kurodai.

Japanese species, Aplysia kurodai, feeds well on Ulva but rejects Gelidium (Geli.) or Pachydictyon (Pach.) with different rhythmic patterned movements of the jaws and radula. During ingestion the jaws open at the radula-protraction phase and remain half open at the initial phase of the radula-retraction, whereas during rejection the jaws open similarly but start to close immediately after the onset of the radula-retraction. These can be induced not only by the natural seaweed but by the extract solutions. We previously showed that the change of the patterned jaw movements from the ingestion to the rejection may result from the decrease in the delay of the firing onset of the jaw-closing (JC) motor neurons during their depolarization. This diminished delay produces a phase advance relative to the radula-retraction phase. In that study, we showed that during ingestion the buccal multiaction (MA) neurons may generate the delay of firing onset of the JC motor neurons by producing monosynaptic inhibitory postsynaptic potentials (IPSPs) during the initial portion of their depolarization. In the present experiments, the firing patterns in the MA neurons induced by application of the Geli. or Pach. extract to the lips were explored in the semi-intact preparations. During the Pach. response the duration and the firing frequency of the MA firing at each depolarizing phase were decreased in comparison with the Ulva response. No decreases in the MA firing were observed during the Geli. response, suggesting that the similar patterned jaw movements during rejection of Geli. and Pach. may be generated by different neural mechanisms. Moreover, the size of the MA-induced IPSP in the JC motor neurons was largely decreased by application of the Geli. or Pach. extract to the lips in the reduced preparations consisting of the tentacle-lips and the cerebral-buccal ganglia. Voltage-clamp experiments on the JC motor neurons showed that the size of synaptic current induced by the MA spikes was decreased by application of these solutions to the lips. The decrease was induced when the buccal ganglia were bathed in a solution to block polysynaptic pathways. These results suggest that the advance of the onset of the JC firing at each depolarizing phase during the Geli. or Pach. response may be mainly or partly caused by the decrease in the size of the MA-induced IPSP in the motor neurons. Modulatory action of cerebral neurons or peripheral afferent neurons in the lip region may contribute to this synaptic plasticity.