6-cyano-7-nitroquinoxaline-2-3-dione and alpha-amino-3-hydroxy-5-tert-butyl-4-isoxazolepropionate

The supraoptic nucleus (SON) contains two types of magnocellular neurosecretory cells: arginine vasopressin (AVP)-producing and oxytocin (OXT)-producing cells. We recently generated and characterised two transgenic rat lines: one expressing an AVP-enhanced green fluorescent protein (eGFP) and the other expressing an OXT-monomeric red fluorescent protein 1 (mRFP1). These transgenic rats enable the visualisation of AVP or OXT neurones in the SON. In the present study, we compared the electrophysiological responses of AVP-eGFP and OXT-mRFP1 neurones to glutamic acid in SON primary cultures. Glutamate mediates fast synaptic transmission through three classes of ionotrophic receptors: the NMDA, AMPA and kainate receptors. We investigated the contributions of the three classes of ionotrophic receptors in glutamate-induced currents. Three different antagonists were used, each predominantly selective for one of the classes of ionotrophic receptor. Next, we focused on the kainate receptors (KARs). We examined the electrophysiological effects of kainic acid (KA) on AVP-eGFP and OXT-mRFP1 neurones. In current clamp mode, KA induced depolarisation and increased firing rates. These KA-induced responses were inhibited by the non-NMDA ionotrophic receptor antagonist 6-cyano-7-nitroquinoxaline-2,3(1H4H)-dione in both AVP-eGFP and OXT-mRFP1 neurones. In voltage clamp mode, the application of KA evoked inward currents in a dose-dependent manner. The KA-induced currents were significantly larger in OXT-mRFP1 neurones than in AVP-eGFP neurones. This significant difference in KA-induced currents was abolished by the GluK1-containing KAR antagonist UBP302. At high concentrations (250-500 μm), the specific GluK1-containing KAR agonist (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) induced significantly larger currents in OXT-mRFP1 neurones than in AVP-eGFP neurones. Furthermore, the difference between the AVP-eGFP and OXT-mRFP1 neurones in the ATPA currents was approximately equal to the difference in the KA currents. These findings suggest that the GluK1-containing KARs may be more highly expressed in OXT neurones than in AVP neurones. These results may provide new insight into the physiology and synaptic plasticity of SON neurones.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Alanine; Animals; Arginine Vasopressin; Cell Separation; Electric Conductivity; Electrophysiology; Excitatory Amino Acid Antagonists; Glutamic Acid; Green Fluorescent Proteins; Isoxazoles; Kainic Acid; Luminescent Proteins; Neurons; Oxytocin; Patch-Clamp Techniques; Primary Cell Culture; Propionates; Rats; Rats, Transgenic; Receptors, Ionotropic Glutamate; Receptors, Kainic Acid; Red Fluorescent Protein; Supraoptic Nucleus; Thymine

Kainate (KA) receptors are expressed widely in the CNS. However, little is known about their functional characterization, molecular identity, and role in synaptic transmission in the forebrain of adult mice. Patch-clamp recordings in genetically modified mice show that postsynaptic KA receptors contribute to fast synaptic transmission in pyramidal neurons in the anterior cingulate cortex (ACC), a forebrain region critical for higher-order cognitive brain functions such as memory and mental disorders. Single-shock stimulation could induce small KA receptor-mediated excitatory postsynaptic currents (KA EPSCs) in the presence of picrotoxin, D-2-amino-5-phosphono-pentanoic acid, and a selective AMPA receptor antagonist, GYKI 53655. KA EPSCs had a significantly slower rise time course and decay time constant compared with AMPA receptor-mediated EPSCs. High-frequency repetitive stimulation significantly facilitated the KA EPSCs. Genetic deletion of the GluR6 or GluR5 subunit significantly reduced, and GluR5 and 6 double knockout completely abolished, KA EPSCs and KA-activated currents in ACC pyramidal neurons. Our results show that KA receptors contribute to synaptic transmission in adult ACC pyramidal neurons and provide a synaptic basis for the physiology and pathology of KA receptors in ACC-related functions.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Benzodiazepines; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GluK2 Kainate Receptor; Gyrus Cinguli; In Vitro Techniques; Isoxazoles; Kainic Acid; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons; Patch-Clamp Techniques; Propionates; Receptors, Kainic Acid; Synaptic Transmission

It has been suggested recently that presynaptic kainate receptors (KARs) are involved in short-term and long-term synaptic plasticity at hippocampal mossy fiber synapses. Using genetic deletion and pharmacology, we here assess the role of GLU(K5) and GLU(K6) in synaptic plasticity at hippocampal mossy fiber synapses. We found that the kainate-induced facilitation was completely abolished in the GLU(K6)-/- mice, whereas it was unaffected in the GLU(K5)-/-. Consistent with this finding, synaptic facilitation was reduced in the GLU(K6)(-/-) and was normal in the GLU(K5)-/-. In agreement with these results and ruling out any compensatory effects in the genetic deletion models, application of the GLU(K5)-specific antagonist LY382884 [(3S,4aR,6S,8aR)-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid] did not affect short-term and long-term synaptic plasticity at the hippocampal mossy fiber synapses. We therefore conclude that the facilitatory effects of kainate on mossy fiber synaptic transmission are mediated by GLU(K6)-containing KARs.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acids, Dicarboxylic; Animals; Benzodiazepines; Cyclopropanes; Excitatory Postsynaptic Potentials; Gene Deletion; GluK2 Kainate Receptor; Glycine; Isoquinolines; Isoxazoles; Kainic Acid; Mice; Mice, Knockout; Mossy Fibers, Hippocampal; Neuronal Plasticity; Patch-Clamp Techniques; Potassium; Propionates; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Kainic Acid; Synaptic Transmission