Juan Lerma

SELECTIVE ANTAGONISM OF AMPA RECEPTORS UNMASKS KAINATE RECEPTOR-MEDIATED RESPONSES IN HIPPOCAMPAL NEURONS

Although both protein and mRNAs for kainate receptor subunits are abundant in several brain regions, the responsiveness of AMPA receptors to kainate has made it difficult to demonstrate the presence of functional kainate-type receptors in native cells. Recently, however, we have shown that many hippocampal neurons in culture express glutamate receptors of the kainate type. The large nondesensitizing response that kainate induces at AMPA receptors precludes detection and analysis of smaller, rapidly desensitizing currents induced by kainate at kainate receptors. Consequently, the functional significance of these strongly desensitizing glutamate receptors remains enigmatic. We report here that the family of new noncompetitive antagonists of AMPA receptors (GYKI 52466 and 53655) minimally affects kainate-induced responses at kainate receptors while completely blocking AMPA receptor-mediated currents, making it possible to separate the responses mediated by each receptor. These compounds will allow determination of the role played by kainate receptors in synaptic transmission and plasticity in the mammalian brain, as well as evaluation of their involvement in neurotoxicity.

Roles and rules of kainate receptors in synaptic transmission.

Functional kainate receptors are ubiquitous in the central nervous system. After a search for their functional significance, a considerable amount of data indicates that this class of glutamate receptors is present at both sides of the synapse. Pre- and postsynaptic kainate receptors can regulate transmission at many synapses in a specific manner, and seem to be involved in short- and long-term plastic phenomena, highlighting their significance for synaptic signalling.

Control of cortical GABA circuitry development by Nrg1 and ErbB4 signalling

Schizophrenia is a complex disorder that interferes with the function of several brain systems required for cognition and normal social behaviour. Although the most notable clinical aspects of the disease only become apparent during late adolescence or early adulthood, many lines of evidence suggest that schizophrenia is a neurodevelopmental disorder with a strong genetic component. Several independent studies have identified neuregulin 1 (NRG1) and its receptor ERBB4 as important risk genes for schizophrenia, although their precise role in the disease process remains unknown. Here we show that Nrg1 and ErbB4 signalling controls the development of inhibitory circuitries in the mammalian cerebral cortex by cell-autonomously regulating the connectivity of specific GABA (γ-aminobutyric acid)-containing interneurons. In contrast to the prevalent view, which supports a role for these genes in the formation and function of excitatory synapses between pyramidal cells, we found that ErbB4 expression in the mouse neocortex and hippocampus is largely confined to certain classes of interneurons. In particular, ErbB4 is expressed by many parvalbumin-expressing chandelier and basket cells, where it localizes to axon terminals and postsynaptic densities receiving glutamatergic input. Gain- and loss-of-function experiments, both in vitro and in vivo, demonstrate that ErbB4 cell-autonomously promotes the formation of axo-axonic inhibitory synapses over pyramidal cells, and that this function is probably mediated by Nrg1. In addition, ErbB4 expression in GABA-containing interneurons regulates the formation of excitatory synapses onto the dendrites of these cells. By contrast, ErbB4 is dispensable for excitatory transmission between pyramidal neurons. Altogether, our results indicate that Nrg1 and ErbB4 signalling is required for the wiring of GABA-mediated circuits in the postnatal cortex, providing a new perspective to the involvement of these genes in the aetiology of schizophrenia.

Kainate Receptors Presynaptically Downregulate GABAergic Inhibition in the Rat Hippocampus

Using microcultured neurons and hippocampal slices, we found that under conditions that completely block AMPA receptors, kainate induces a reduction in the effectiveness of GABAergic synaptic inhibition. Evoked inhibitory postsynaptic currents (IPSCs) were decreased by kainate by up to 90%, showing a bell-shaped dose-response curve similar to that of native kainate-selective receptors. The down-regulation of GABAergic inhibition was not affected by antagonism of metabotropic receptors, while it was attenuated by CNQX. Kainate increased synaptic failures and reduced the frequency of miniature IPSCs, indicating a presynaptic locus of action. In vivo experiments using brain dialysis demonstrated that kainate reversibly abolished recurrent inhibition and induced an epileptic-like electroencephalogram (EEG) activity. These results indicate that kainate receptor activation down-regulates GABAergic inhibition by modulating the reliability of GABA synapses.

Kainate Receptor Modulation of GABA Release Involves a Metabotropic Function

The mechanism through which kainate receptors downregulate the release of GABA in the hippocampus is not known. We have found that the action of kainate on the hippocampal inhibitory postsynaptic current (IPSC) is mediated by a metabotropic process that is sensitive to Pertussis toxin (PTx) and independent of ion channel current. The downregulation of GABA IPSCs by kainate was also prevented in a dose-dependent manner by calphostin C, a specific inhibitor of PKC, and the inhibition of phospholipase C (PLC) drastically reduced the action of kainate. The effect of kainate was completely occluded by phorbol esters and by increasing extracellular Ca2+ but remained unaltered after inhibition or activation of protein kinase A (PKA). These results demonstrate that the activation of kainate receptors triggers a second messenger cascade, which results in the stimulation of PKC, and therefore document a metabotropic action of kainate receptors, which results in the inhibition of GABA release.

Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor.

This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate‐induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells. Exposure (30 min) of energy deprived cells to L‐glutamate (1–100 μM) caused a concentration‐dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L‐glutamate‐induced neurotoxicity (EC50=5 μM) was blocked by the specific NMDA receptor antagonist MK‐801 (dizocilpine). Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 μM (EC100) L‐glutamate with the rank order (EC50 in μM): antazoline (13)>cirazoline (44)>LSL 61122 [2‐styryl‐2‐imidazoline] (54)>LSL 60101 [2‐(2‐benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2‐(1,4‐benzodioxan‐6‐yl)‐4,5‐dihydroimidazole] (101)>RX821002 (2‐methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors. Imidazol(ine) drugs and agmatine fully inhibited [3H]‐(+)‐MK‐801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L‐glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]‐(+)‐MK‐801 binding. In HEK‐293 cells transfected to express the NR1‐1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage‐ and concentration‐dependent block of glutamate‐induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10–12 μM at 0 mV. It is concluded that imidazol(ine) drugs and agmatine are neuroprotective against glutamate‐induced necrotic neuronal cell death in vitro and that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore.

Glutamate receptors of the kainate type and synaptic transmission

Glutamic acid is an important excitatory neurotransmitter in the mammalian CNS. It has been established that synaptic transmission is mediated mostly by the ionotropic glutamate receptors AMPA and NMDA, with fast and slow kinetics, respectively. The recent demonstration in hippocampal neurones of a class of glutamate receptors that are activated by kainate and not by AMPA (that is, kainate-selective receptors) opens the possibility that receptors, others than those of the AMPA type, might also be involved in fast neurotransmission. The lack of specific pharmacological tools to dissect out AMPA from kainate receptors has hampered the functional study of kainate receptors. However, the recent finding that a 2,3-benzodiazepine (GYK153655) behaves as a selective antagonist of AMPA receptors allows us to address the question of the role of rapidly inactivating kainate receptors in synaptic transmission.

Four Residues of the Extracellular N-Terminal Domain of the NR2A Subunit Control High-Affinity Zn2+ Binding to NMDA Receptors

NMDA receptors are allosterically inhibited by Zn2+ ions in a voltage-independent manner. The apparent affinity for Zn2+ of the heteromeric NMDA receptors is determined by the subtype of NR2 subunit expressed, with NR2A-containing receptors being the most sensitive (IC50, approximately 20 nM) and NR2C-containing receptors being the least sensitive (IC50, approximately 30 microM). Using chimeras constructed from these two NR2 subtypes, we show that the N-terminal LIVBP-like domain of the NR2A subunit controls the high-affinity Zn2+ inhibition. Mutations at four residues in this domain markedly reduce Zn2+ affinity (by up to >500-fold) without affecting either receptor activation by glutamate and glycine or inhibition by extracellular protons and Ni2+ ions, indicating that these residues most likely participate in high-affinity Zn2+ binding.

The Synaptic Hypothesis of Schizophrenia

12 paginas, 5 figuras.-- The following is a report of the meeting “Synaptic Dysfunction and Schizophrenia” held at the Juan March Center for International Meetings on Biology Workshop in Madrid, Spain, 10–12 February 2003.

Kainate Receptor Subunits Expressed in Single Cultured Hippocampal Neurons: Molecular and Functional Variants by RNA Editing

To determine the kainate receptor subunits that are found in native kainate receptors, we have applied a multiplex PCR of cDNAs reverse transcribed from mRNA harvested from single cultured hippocampal neurons after electrophysiological recording. We found that all the cells showing rapidly desensitizing currents in response to kainate express the GluR6 subunit mRNA, and that some of them also express the GluR5 subunit mRNA. No GluR7, KA-1, or KA-2 subunit mRNAs were detected. Analysis of the editing sites of the GluR6 mRNA demonstrated that the three editing sites present in these subunits are edited to a different extent. Predominant expression of the unedited variant (Q) was observed, but edited and unedited variants may coexist in the same cell. In addition, we show that the Q/R site from the GluR6 subunit controls functional properties of native kainate receptors.