NEUROPHARMACOLOGY NEUROREPORT 0959-4965 & Lippincott Williams & Wilkins Vol 11 No 12 21 August 2000 2695 Co-existence in DUM neurones of two GluCl channels that differ in their picrotoxin sensitivity Vale Ârie Raymond, 1,2 David B. Sattelle 2 and Bruno Lapied 1,CA 1 Laboratoire de Neurophysiologie, UPRES EA 2647, Universite  d'Angers, UFR Sciences, 2 Bd Lavoisier, F-49045, Angers Cedex, France; 2 MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK CA Corresponding Author Received 17 May 2000; accepted 9 June 2000 Whole cell patch-clamp electrophysiology was used to study the effects of L-glutamate on dissociated cockroach ( Periplaneta americana) dorsal unpaired median (DUM) neurones. Applica- tion of L-glutamate via pressure-ejection pipette resulted in a two-component hyperpolarization, consisting of an initial transient and a second, prolonged phase. Under voltage-clamp, using isotonic chloride in the saline and intrapipette solutions, two L-glutamate-gated inward currents were characterized. Their reversal potentials were close to the equilibrium potential for chloride ions. One component was selectively activated by ibotenate and was sensitive to picrotoxin (100 ìM), BIDN (10 ìM) and the phenylpyrazole insecticide ®pronil (10 ìM), known to be potent blockers of insect GABA- gated chloride channels. The second component was insensitive to picrotoxin (100 ìM) and BIDN (10 ìM). These ®ndings demonstrated, for the ®rst time, the co-existence of two pharmacologically distinct native insect neuronal L-glutamate- gated chloride channels. NeuroReport 11:2695±2701 & 2000 Lippincott Williams & Wilkins. Key words: BIDN; Fipronil; L-glutamate-gated chloride channels; Insect neurones; Picrotoxin INTRODUCTION The neurotransmitter actions of L-glutamate are mediated by two distinct classes of receptors referred to as ionotropic [1,2] and metabotropic [1,3]. All known vertebrate ionotro- pic L-glutamate receptors gate cation channels and are classi®ed into three subtypes: AMPA, kainate (KA) and NMDA receptors. AMPA and KA receptors gate channels permeable mainly to sodium and potassium ions [1]. The NMDA receptors gate ion channels permeable to calcium as well as sodium and potassium. They also show voltage- dependent block by magnesium ions [4]. L-Glutamate- gated cation channels are implicated in learning and memory, and in neurodegenerative diseases such as Alz- heimer's disease, Huntington's disease, amyotrophic lateral sclerosis and epilepsy [2,5]. There is also considerable evidence that in insects L-glutamate-gated cation channels mediate chemical neurotransmission at the neuromuscular junction [6]. However, another class of ionotropic L-gluta- mate receptors gate chloride channels (GluCls) and, to date, they have been found only in invertebrates [7±9]. They are important sites of action of endectocides such as ivermectin. In contrast to the well-studied ionotropic L- glutamate receptors of vertebrates, much less is known of the subtypes and their functions in the case of invertebrate and particularly in insect neuronal preparations. Although some qualitative electrophysiological properties of neuro- nal ionotropic glutamate receptors have been established [7], the number, subtypes, distributions and physiological roles of these receptors in insect neurones still remain unclear. This is reinforced by previous electrophysiological investigations performed in insect neurones that have reported several complex distinct L-glutamate-induced re- sponses suggesting the existence of more than one type of L-glutamate receptors [7,10,11]. These include a GluCl activated by both L-glutamate and ibotenate and blocked by picrotoxin (PTX) and insensitive to CNQX; a L-gluta- mate-gated cationic receptor insensitive to PTX and blocked by CNQX; an ibotenate-induced hyperpolarization (through a chloride channel) insensitive to L-glutamate and CNQX; and a quisqualate-induced slow depolarization insensitive to both PTX and CNQX. Furthermore, on the same neurone, a biphasic response to L-glutamate are sometimes detected, an initial rapid hyperpolarization followed by a slow depolarization which is mimicked by quisqualate and kainate [7]. Dorsal unpaired median (DUM) neurones in the cockroach thoracic ganglion can also respond to L-glutamate with either a hyperpolarization or a depolarization both of which are sensitive to PTX. These responses, observed at resting membrane potentials, are accompanied by an increase in membrane conductance [12] and the two components appeared to be attributable to the shift of the chloride equilibrium potential during the