RESUMEN
Our understanding of the molecular properties of kainate receptors and their involvement in synaptic physiology has progressed significantly over the last 30 years. A plethora of studies indicate that kainate receptors are important mediators of the pre- and postsynaptic actions of glutamate, although the mechanisms underlying such effects are still often a topic for discussion. Three clear fields related to their behavior have emerged: there are a number of interacting proteins that pace the properties of kainate receptors; their activity is unconventional since they can also signal through G proteins, behaving like metabotropic receptors; they seem to be linked to some devastating brain diseases. Despite the significant progress in their importance in brain function, kainate receptors remain somewhat puzzling. Here we examine discoveries linking these receptors to physiology and their probable implications in disease, in particular mood disorders, and propose some ideas to obtain a deeper understanding of these intriguing proteins.
Asunto(s)
Enfermedades del Sistema Nervioso Central/fisiopatología , Salud , Trastornos Mentales/fisiopatología , Receptores de Ácido Kaínico/metabolismo , Receptores de Ácido Kaínico/fisiología , Transmisión Sináptica/fisiología , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Encéfalo/fisiología , Enfermedades del Sistema Nervioso Central/genética , Ácido Glutámico/fisiología , Humanos , Modelos Neurológicos , Unión Proteica/fisiologíaRESUMEN
We have investigated the mechanisms underlying the facilitatory modulation mediated by kainate receptor (KAR) activation in the cortex, using isolated nerve terminals (synaptosomes) and slice preparations. In cortical nerve terminals, kainate (KA, 100 µM) produced an increase in 4-aminopyridine (4-AP)-evoked glutamate release. In thalamocortical slices, KA (1 µM) produced an increase in the amplitude of evoked excitatory post-synaptic currents (eEPSCs) at synapses established between thalamic axon terminals from the ventrobasal nucleus onto stellate neurons of L4 of the somatosensory cortex. In both, synaptosomes and slices, the effect of KA was antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione, and persisted after pre-treatment with a cocktail of antagonists of other receptors whose activation could potentially have produced facilitation of release indirectly. Mechanistically, the observed effects of KA appear to be congruent in synaptosomal and slice preparations. Thus, the facilitation by KA of synaptosomal glutamate release and thalamocortical synaptic transmission were suppressed by the inhibition of protein kinase A and occluded by the stimulation of adenylyl cyclase. Dissecting this G-protein-independent regulation further in thalamocortical slices, the KAR-mediated facilitation of synaptic transmission was found to be sensitive to the block of Ca(2+) permeant KARs by philanthotoxin. Intriguingly, the synaptic facilitation was abrogated by depletion of intracellular Ca(2+) stores by thapsigargin, or inhibition of Ca(2+) -induced Ca(2+) -release by ryanodine. Thus, the KA-mediated modulation was contingent on both Ca(2+) entry through Ca(2+) -permeable KARs and liberation of intracellular Ca(2+) stores. Finally, sensitivity to W-7 indicated that the increased cytosolic [Ca(2+) ] underpinning KAR-mediated regulation of synaptic transmission at thalamocortical synapses, requires downstream activation of calmodulin. We conclude that neocortical pre-synaptic KARs mediate the facilitation of glutamate release and synaptic transmission by a Ca(2+) -calmodulin dependent activation of an adenylyl cyclase/cAMP/protein kinase A signalling cascade, independent of G-protein involvement.
Asunto(s)
Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/fisiología , Corteza Cerebral/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Glutamatos/metabolismo , Receptores de Ácido Kaínico/fisiología , Receptores Presinapticos/fisiología , Sinapsis/fisiología , Tálamo/fisiología , Algoritmos , Animales , Corteza Cerebral/efectos de los fármacos , AMP Cíclico/metabolismo , Interpretación Estadística de Datos , Fenómenos Electrofisiológicos , Agonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/fisiología , Técnicas In Vitro , Ácido Kaínico/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Técnicas de Placa-Clamp , Receptores de Ácido Kaínico/efectos de los fármacos , Receptores Presinapticos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sinaptosomas/metabolismo , Tálamo/efectos de los fármacosRESUMEN
Long-term synaptic enhancements in cortical and thalamic auditory inputs to the lateral nucleus of the amygdala (LAn) mediate encoding of conditioned fear memory. It is not known, however, whether the convergent auditory conditioned stimulus (CSa) pathways interact with each other to produce changes in their synaptic function. We found that continuous paired stimulation of thalamic and cortical auditory inputs to the LAn with the interstimulus delay approximately mimicking a temporal pattern of their activation in behaving animals during auditory fear conditioning resulted in persistent potentiation of synaptic transmission in the cortico-amygdala pathway in rat brain slices. This form of input timing-dependent plasticity (ITDP) in cortical input depends on inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) release from internal stores and postsynaptic Ca(2+) influx through calcium-permeable kainate receptors during its induction. ITDP in the auditory projections to the LAn, determined by characteristics of presynaptic activity patterns, may contribute to the encoding of the complex CSa.
Asunto(s)
Amígdala del Cerebelo/fisiología , Corteza Cerebral/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Tálamo/fisiología , Animales , Señalización del Calcio/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Inositol 1,4,5-Trifosfato/metabolismo , Vías Nerviosas/fisiología , Ratas , Receptores de Ácido Kaínico/fisiología , Receptores de Glutamato Metabotrópico/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Presynaptic kainate receptors play an important role in synaptic transmission and short-term plasticity to profoundly regulate network activity in many parts of the mammalian brain. In primary sensory neocortex, where short-term synaptic plasticity is important for receptive field structure and information processing, kainate receptors are highly expressed and regulate thalamocortical inputs, particularly during development. However, the mechanisms of the kainate receptor-dependent presynaptic regulation of thalamocortical transmission are unclear. We therefore investigated this issue using electrophysiology in neonatal thalamocortical slices of barrel cortex combined with pharmacology and biochemical analyses. We show that presynaptic kainate receptors can both facilitate or depress synaptic transmission depending on the extent of their activation. This bi-directional regulation is mediated in part by kainate receptors that directly influence thalamocortical axonal excitability, but also likely involves receptors acting at thalamocortical terminals to regulate transmitter release. The efficacy of kainate in regulating thalamocortical transmission is low compared to that reported for other inputs. Consistent with this low efficacy, our biochemical analyses indicate that the presynaptic kainate receptors regulating neonatal thalamocortical inputs likely lack the high kainate affinity GluK4 and 5 subunits. Thus thalamocortical transmission can be bi-directionally regulated by low affinity kainate receptors through two mechanisms. Such presynaptic regulation provides a potentially powerful mechanism to influence sensory processing during development of barrel cortex.
Asunto(s)
Receptores de Ácido Kaínico/fisiología , Receptores Presinapticos/fisiología , Corteza Somatosensorial/fisiología , Transmisión Sináptica/fisiología , Tálamo/fisiología , Animales , Animales Recién Nacidos , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Ácido Kaínico/farmacología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/fisiología , Técnicas de Placa-Clamp , Receptores de Ácido Kaínico/biosíntesis , Corteza Somatosensorial/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacosRESUMEN
Corticothalamic fibres, which originate from layer VI pyramidal neurons in the cerebral cortex, provide excitatory synaptic inputs to both thalamic relay neurons and reticular neurons; reticular neurons in turn supply inhibitory inputs to thalamic relay neurons. Pyramidal cells in layer VI in the mouse somatosensory cortex highly express mRNA encoding kainate receptors, which facilitate or depress transmitter release at several synapses in the central nervous system. We report here that contrary modulation of transmitter release from corticothalamic fibres onto thalamic relay and reticular neurons is mediated by activation of kainate receptors in mouse thalamic ventrobasal complex and thalamic reticular nucleus. Exogenous kainate presynaptically depresses the synaptic transmission at corticothalamic synapses onto thalamic relay neurons, but facilitates it at corticothalamic synapses onto reticular neurons. Meanwhile, the lemniscal synaptic transmission, which sends primary somatosensory inputs to relay neurons, is not affected by kainate. In addition, GluR5-containing kainate receptors are involved in the depression of corticothalamic synaptic transmission onto relay neurons, but not onto reticular neurons. Furthermore, synaptically activated kainate receptors mimic these effects; high-frequency stimulation of corticothalamic fibres depresses synaptic transmission onto relay neurons, but facilitates it onto reticular neurons. Our results suggest that the opposite sensitivity of kainate receptors at the two corticothalamic synapses is governed by cortical activity and regulates the balance of excitatory and inhibitory inputs to thalamic relay neurons and therefore their excitability.
Asunto(s)
Corteza Cerebral/metabolismo , Neuronas/metabolismo , Neurotransmisores/metabolismo , Receptores de Ácido Kaínico/fisiología , Sinapsis/metabolismo , Tálamo/metabolismo , Animales , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Sex differences in brain morphology underlie physiological and behavioral differences between males and females. During the critical perinatal period for sexual differentiation in the rat, gonadal steroids act in a regionally specific manner to alter neuronal morphology. Using Golgi-Cox impregnation, we examined several parameters of neuronal morphology in postnatal day 2 (PN2) rats. We found that in the ventromedial nucleus of the hypothalamus (VMN) and in areas just dorsal and just lateral to the VMN that there was a sex difference in total dendritic spine number (males greater) that was abolished by treating female neonates with exogenous testosterone. Dendritic branching was similarly sexually differentiated and hormonally modulated in the VMN and dorsal to the VMN. We then used spinophilin, a protein that positively correlates with the amount of dendritic spines, to investigate the mechanisms underlying these sex differences. Estradiol, which mediates most aspects of masculinization and is the aromatized product of testosterone, increased spinophilin levels in female PN2 rats to that of males. Muscimol, an agonist at GABA(A) receptors, did not affect spinophilin protein levels in either male or female neonates. Kainic acid, an agonist at glutamatergic AMPA/kainate receptors, mimicked the effect of estradiol in females. Antagonizing AMPA/kainate receptors with NBQX prevented the estradiol-induced increase in spinophilin in females but did not affect spinophilin level in males.
Asunto(s)
Andrógenos/farmacología , Hipotálamo/efectos de los fármacos , Receptores AMPA/fisiología , Receptores de Ácido Kaínico/fisiología , Caracteres Sexuales , Testosterona/farmacología , Animales , Animales Recién Nacidos , Dendritas/ultraestructura , Interacciones Farmacológicas , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Hipotálamo/citología , Ácido Kaínico/farmacología , Masculino , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/ultraestructura , Embarazo , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/fisiología , Tinción con Nitrato de Plata/métodosRESUMEN
During the first week of life, there is a shift from kainate to AMPA receptor-mediated thalamocortical transmission in layer IV barrel cortex. However, the mechanisms underlying this change and the differential properties of AMPA and kainate receptor-mediated transmission remain essentially unexplored. To investigate this, we studied the quantal properties of AMPA and kainate receptor-mediated transmission using strontium-evoked miniature EPSCs. AMPA and kainate receptor-mediated transmission exhibited very different quantal properties but were never coactivated by a single quantum of transmitter, indicating complete segregation to different synapses within the thalamocortical input. Nonstationary fluctuation analysis showed that synaptic AMPA receptors exhibited a range of single-channel conductance (gamma) and a strong negative correlation between gamma and functional channel number, indicating that these two parameters are reciprocally regulated at thalamocortical synapses. We obtained the first estimate of gamma for synaptic kainate receptors (<2 pS), and this primarily accounted for the small quantal size of kainate receptor-mediated transmission. Developmentally, the quantal contribution to transmission of AMPA receptors increased and that of kainate receptors decreased. No changes in AMPA or kainate quantal amplitude or in AMPA receptor gamma were observed, demonstrating that the developmental change was attributable to a decrease in the number of kainate synapses and an increase in the number of AMPA synapses contributing to transmission. Therefore, we demonstrate fundamental differences in the quantal properties for these two types of synapse. Thus, the developmental switch in transmission will dramatically alter information transfer at thalamocortical inputs to layer IV.
Asunto(s)
Corteza Cerebral/citología , Receptores AMPA/fisiología , Receptores de Ácido Kaínico/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Tálamo/citología , Animales , Animales Recién Nacidos , Ácido Aspártico/farmacología , Calcio/farmacología , Corteza Cerebral/crecimiento & desarrollo , Quelantes/farmacología , Relación Dosis-Respuesta en la Radiación , Interacciones Farmacológicas , Ácido Egtácico/farmacología , Estimulación Eléctrica/métodos , Agonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Excitadores/efectos de la radiación , Técnicas In Vitro , Ácido Kaínico/farmacología , Ratones , Modelos Neurológicos , Método de Montecarlo , Conducción Nerviosa/efectos de los fármacos , Conducción Nerviosa/fisiología , Conducción Nerviosa/efectos de la radiación , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/fisiología , Neuronas/citología , Técnicas de Placa-Clamp/métodos , Estroncio/farmacología , Tálamo/crecimiento & desarrollo , Factores de Tiempo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Although the interactions between sex steroids and GnRH have been extensively studied, little is known about the mechanism of estradiol (E2) effects on GnRH secretion. In the present study, we used retrochiasmatic hypothalamic explants of 50-d-old male rats, and we observed that E2 significantly increased the glutamate-evoked GnRH secretion in vitro within 15 min in a dose-dependent manner. E2 also significantly increased the L-arginine-evoked GnRH secretion. E2 effects were time dependent because the initially ineffective 10(-9) M concentration became effective after 5 h of incubation. The E2 effects involved the estrogen receptor (ER) alpha because they were similarly obtained with the specific ER alpha agonist 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole. The use of glutamate receptor agonists and antagonists indicated that E2 effects on GnRH secretion evoked by both glutamate and L-arginine involved the 2-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid/kainate receptors. Similar E2 effects on the kainate-evoked secretion were observed throughout development in both sexes. The observation of similar E2 effects using explants containing the median eminence alone indicated that the median eminence was a direct target for E2 rapid effects on the glutamate-evoked GnRH secretion. The signaling pathways involved in E2 effects included an increase in intracellular calcium and the activation of protein kinase A, protein kinase C, and MAPK. It is concluded that E2 can stimulate the glutamate- and nitric oxide-evoked GnRH secretion in vitro through a rapid pathway involving the ER and kainate receptor as well as through a slower mechanism responding to lower E2 concentrations.
Asunto(s)
Estradiol/farmacología , Hormona Liberadora de Gonadotropina/metabolismo , Hipotálamo/metabolismo , Receptores de Estrógenos/fisiología , Receptores de Ácido Kaínico/fisiología , Transducción de Señal/fisiología , Envejecimiento , Animales , Arginina/farmacología , Receptor alfa de Estrógeno/agonistas , Receptor alfa de Estrógeno/fisiología , Femenino , Ácido Glutámico/farmacología , Hipotálamo/efectos de los fármacos , Cinética , Masculino , Eminencia Media/efectos de los fármacos , Eminencia Media/metabolismo , Óxido Nítrico/farmacología , Área Preóptica/efectos de los fármacos , Área Preóptica/metabolismo , Ratas , Ratas Wistar , Receptores de Glutamato/efectos de los fármacos , Receptores de Glutamato/fisiología , Receptores de Ácido Kaínico/efectos de los fármacosRESUMEN
Intrathecal pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists blocks development of spinal sensitization in a number of pain models. In contrast, secondary mechanical allodynia evoked by thermal injury (52.5 degrees C for 45 s) applied to the hind paw of the rat is not blocked by intrathecal pretreatment with NMDA receptor antagonists. It is, however, blocked by antagonists to the non-NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/KA) and calcium-permeable AMPA/KA receptors. These findings suggest a role for these receptors in the development of spinal sensitization. The present study used the same thermal injury model to assess the effects of the AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and specific calcium-permeable AMPA/KA receptor antagonists philanthotoxin (PHTx) and joro spider toxin (JST) when given as postinjury treatments. Intrathecal saline injection at 5 and 30 min postinjury had no effect on thermal injury-evoked allodynia as measured by calibrated von Frey filaments. In contrast, 36 nmol of CNQX given at either time point reversed allodynia. Intrathecal 13 nmol of PHTx or 9 nmol of JST (higher doses than that required for pretreatment) reversed allodynia at the 5-min time point, but neither drug was antiallodynic at the 30-min time point. Thus, secondary mechanical allodynia in this model is not maintained by calcium-permeable AMPA/KA receptors, but instead requires activation of calcium-impermeable AMPA/KA receptors. This finding supports a role for AMPA/KA receptor function in responses occurring during spinal sensitization.
Asunto(s)
Quemaduras/fisiopatología , Calcio/metabolismo , Dolor/fisiopatología , Receptores AMPA/fisiología , Receptores de Ácido Kaínico/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Modelos Animales de Enfermedad , Antagonistas de Aminoácidos Excitadores/farmacología , Inyecciones Espinales , Ácido Kaínico/farmacología , Corteza Motora , Umbral del Dolor , Ratas , Venenos de Araña/farmacología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
We established a model of cortical development that arrests the birth of layer 4 cells by injecting methylazoxymethanol (MAM) on embryonic day 33 (E33) in ferrets. This leads to adult somatosensory cortex with a very thin layer 4. Earlier, we determined the relative absence of layer 4 changed the growth and differentiation of the somatosensory cortex and the growth of thalamic afferents into the cortical plate. To identify other features of cortical organization that might be altered after MAM treatment, we assessed the distribution of selected excitatory and inhibitory receptors in area 3b of ferret somatosensory cortex. Initial screening revealed the distribution of several excitatory receptors (NMDA, AMPA, kainate) in E33 MAM-treated cortex was similar to that in normal adult animals. In contrast, the binding pattern of inhibitory GABAA receptors was altered in MAM-treated cortex. Normally, GABAA receptors densely locate in central layers of cortex. In E33 MAM-treated animals, GABAA receptor binding extended superficially, covering a broader area of cortex. Further experiments using antibodies directed against GABAAalpha receptors disclosed that pan alpha GABAA receptors strongly localize to layer 4 in normal area 3b. In E33 MAM-treated cortex, however, GABAAalpha receptors extend outside and are located above and below the very thin layer 4. The redistribution of inhibitory receptors suggests that layer 4 plays an important role in regulating thalamic terminations and also in the resulting ability to refine processing of incoming stimuli.
Asunto(s)
Hurones/fisiología , Acetato de Metilazoximetanol/análogos & derivados , Acetato de Metilazoximetanol/toxicidad , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/fisiología , Corteza Somatosensorial/efectos de los fármacos , Corteza Somatosensorial/fisiología , Animales , Autorradiografía , Femenino , Inmunohistoquímica , Fibras Nerviosas/fisiología , Neuronas Aferentes/fisiología , Embarazo , Receptores AMPA/fisiología , Receptores de Ácido Kaínico/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Corteza Somatosensorial/citología , Tálamo/citología , Tálamo/fisiologíaRESUMEN
The mitochondrion has moved to the center stage in the drama of the life and death of the neuron. The mitochondrial membrane potential controls the ability of the organelle to generate ATP, generate reactive oxygen species and sequester Ca(2+) entering the cell. Each of these processes interact, and their deconvolution is far from trivial. The cultured cerebellar granule cell provides a model in which knowledge gained from studies on isolated mitochondria can be applied to study the role played by the organelles in the maintenance of Ca(2+) homeostasis in the cell under resting, stimulated and pathophysiological conditions. In particular, mitochondria play a complex role in the response of the neuron to excitotoxic stimulation of NMDA and AMPA-kainate selective glutamate receptors. One goal of research in this area is to provide clues as to possible ways in which modulators of mitochondrial function may be used as neuroprotective agents, since mitochondrial Ca(2+) accumulation seems to play a key role in glutamate excitotoxicity.
Asunto(s)
Calcio/metabolismo , Metabolismo Energético/fisiología , Mitocondrias/fisiología , Neuronas/fisiología , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico/fisiología , Muerte Celular/fisiología , Células Cultivadas , Cerebelo/citología , Cerebelo/metabolismo , Citoplasma/metabolismo , Ácido Glutámico/metabolismo , Humanos , Potenciales de la Membrana/fisiología , Oligomicinas/farmacología , Fosforilación Oxidativa/efectos de los fármacos , Ácido Pirúvico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Ácido Kaínico/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Desacopladores/farmacologíaRESUMEN
Kainate receptors have been studied extensively in vitro, but how they might function physiologically remains unclear. We studied kainate receptor modulation of synaptic responses in the rat ventrobasal thalamus using the novel antagonist LY382884 and the agonist ATPA (selective for GluR5-containing kainate receptors) as tools. No evidence could be found for a direct contribution of kainate receptors to responses of thalamic relay cells to lemniscal (sensory) input in thalamic slices studied with the aid of intracellular and field potential recordings, using selective AMPA and NMDA receptor antagonists and LY382884. However, the GluR5 agonist ATPA reduced the IPSPs originating from the thalamic reticular nucleus. Extracellular single-neurone recordings in anaesthetised rats showed that excitatory responses evoked by physiological vibrissa afferent stimulation were reduced by LY382884 applied iontophoretically at the recording site. This action of the antagonist was occluded when GABA receptors were blocked, indicating that the reduction in excitatory sensory responses by LY382884 is due to an action on GABAergic inhibition arising from the thalamic reticular nucleus. Further experiments showed that these actions depended on whether inhibition was evoked during activation of the excitatory receptive field rather than when inhibition was evoked from a surround vibrissa. We suggest that GluR5 is located presynaptically on inhibitory GABAergic terminals of thalamic reticular nucleus neurones, and that it is normally activated by glutamate spillover from synapses between excitatory afferents and relay neurones during physiological stimulation. We propose that this GluR5-activated disinhibition has an important novel role in extracting sensory information from background noise.
Asunto(s)
Receptores de Ácido Kaínico/fisiología , Sensación/fisiología , Tálamo/fisiología , Animales , Electrofisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Antagonistas del GABA/farmacología , Ácido Glutámico/fisiología , Técnicas In Vitro , Iontoforesis , Isoquinolinas/farmacología , Masculino , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Estimulación Física , Ratas , Receptores de Ácido Kaínico/antagonistas & inhibidores , Sinapsis/fisiología , Transmisión Sináptica/efectos de los fármacos , Vibrisas/fisiología , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Kainate (KA) receptor-mediated excitatory postsynaptic currents (EPSCs) exhibit slow kinetics at the great majority of synapses. However, native or heterologously expressed KA receptors exhibit rapid kinetics in response to agonist application. One possibility to explain this discrepancy is that KA receptors are extrasynaptic and sense glutamate diffusing from the synaptic cleft. We investigated this by studying the effect of three manipulations that change glutamate clearance on evoked KA EPSCs at thalamocortical synapses. First, we used high-frequency stimulation to increase extrasynaptic glutamate levels. This caused an apparent increase in the relative contribution of the KA EPSC to transmission and slowed the decay kinetics. However, scaling and summing the EPSC evoked at low frequency reproduced this, demonstrating that the effect was due to postsynaptic summation of KA EPSCs. Second, we applied inhibitors of high-affinity glutamate transport. This caused a depression in both AMPA and KA EPSC amplitude due to the activation of a presynaptic glutamatergic autoreceptor. However, transport inhibitors had no selective effect on the amplitude or kinetics of the KA EPSC. Third, to increase glutamate clearance, we raised temperature during recordings. This shortened the decay of both the AMPA and KA components and increased their amplitudes, but this effect was the same for both. Therefore these data provide evidence against glutamate diffusion out of the synaptic cleft as the mechanism for the slow kinetics of KA EPSCs. Other possibilities such as interactions of KA receptors with other proteins or novel properties of native synaptic heteromeric receptors are required to explain the slow kinetics.
Asunto(s)
Corteza Cerebral/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Ácido Glutámico/metabolismo , Ácido Kaínico/análogos & derivados , Receptores de Ácido Kaínico/fisiología , Sinapsis/metabolismo , Tálamo/fisiología , 2-Amino-5-fosfonovalerato/farmacología , Animales , Ácido Aspártico/análogos & derivados , Ácido Aspártico/farmacología , Estimulantes del Sistema Nervioso Central/farmacología , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Ácido Kaínico/farmacología , Cinética , Técnicas de Cultivo de Órganos , Picrotoxina/farmacología , Ratas , Ratas Wistar , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , TemperaturaRESUMEN
Previous work showed that the dorsal periaqueductal gray is involved in the inhibition of fear-potentiated startle. The present study investigated the effects of blockade and stimulation of Kainate/AMPA and GABA(A) receptors within the dorsal periaqueductal gray on expression and conditioned inhibition of fear-potentiated startle. Blockade of the Kainate/AMPA receptors enhanced whereas stimulation of the Kainate/AMPA receptors decreased expression of fear-potentiated startle. These effects do not reflect conditioned inhibition since this modulation was not changed by injections of Kainate/AMPA receptor agonists or antagonists into the dorsal periaqueductal gray. Stimulation and blockade of GABA(A) receptors within the dorsal periaqueductal gray neither affected expression of fear-potentiated startle nor its conditioned inhibition. The present results together with findings from the literature indicate that glutamate in the dorsal periaqueductal gray is a critical substrate for the expression and modulation of fear-related behaviours.
Asunto(s)
Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Miedo/fisiología , Sustancia Gris Periacueductal/fisiología , Receptores AMPA/fisiología , Receptores de GABA-A/fisiología , Receptores de Ácido Kaínico/fisiología , Reflejo de Sobresalto/fisiología , Estimulación Acústica , Animales , Condicionamiento Clásico , Electrochoque , Agonistas de Aminoácidos Excitadores/administración & dosificación , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Ácido Kaínico/administración & dosificación , Ácido Kaínico/farmacología , Luz , Masculino , Microinyecciones , Sustancia Gris Periacueductal/efectos de los fármacos , Picrotoxina/farmacología , Piperidinas/administración & dosificación , Piperidinas/farmacología , Quinoxalinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
To investigate synaptic mechanisms underlying information processing in auditory cortex, we examined cholinergic modulation of synaptic transmission in a novel slice preparation containing thalamocortical and intracortical inputs to mouse auditory cortex. Extracellular and intracellular recordings were made in cortical layer IV while alternately stimulating thalamocortical afferents (via medial geniculate or downstream subcortical stimulation) and intracortical afferents. Either subcortical or intracortical stimulation elicited a fast, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-sensitive, monosynaptic EPSP followed by long-duration, polysynaptic activity. The cholinergic agonist carbachol suppressed each of the synaptic potentials to different degrees. At low concentrations (5 microM) carbachol strongly reduced (>60%) the polysynaptic slow potentials for both pathways but did not affect the monosynaptic fast potentials. At higher doses (10-50 microM), carbachol also reduced the fast potentials, but reduced the intracortically-elicited fast potential significantly more than the thalamocortically-elicited fast potential, which at times was actually enhanced. Atropine (0.5 microM) blocked the effects of carbachol, indicating muscarinic receptor involvement. We conclude that muscarinic modulation can strongly suppress intracortical synaptic activity while exerting less suppression, or actually enhancing, thalamocortical inputs. Such differential actions imply that auditory information processing may favor sensory information relayed through the thalamus over ongoing cortical activity during periods of increased acetylcholine (ACh) release.
Asunto(s)
2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Corteza Auditiva/fisiología , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Transmisión Sináptica/efectos de los fármacos , Tálamo/fisiología , Animales , Atropina/farmacología , Corteza Auditiva/efectos de los fármacos , Estimulación Eléctrica , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Técnicas In Vitro , Ratones , Ratones Endogámicos , Técnicas de Placa-Clamp , Receptores AMPA/fisiología , Receptores de Ácido Kaínico/fisiología , Transmisión Sináptica/fisiología , Tálamo/efectos de los fármacosRESUMEN
Most of the fast excitatory synaptic transmission in the mammalian brain is mediated by ionotrophic glutamate receptors, of which there are three subtypes: AMPA (alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate), NMDA (N-methyl-D-aspartate) and kainate. Although kainate-receptor subunits (GluR5-7, KA1 and 2) are widely expressed in the mammalian central nervous system, little is known about their function. The development of pharmacological agents that distinguish between AMPA and kainate receptors has now allowed the functions of kainate receptors to be investigated. The modulation of synaptic transmission by kainate receptors and their synaptic activation in a variety of brain regions have been reported. The expression of kainate receptor subunits is developmentally regulated but their role in plasticity and development is unknown. Here we show that developing thalamocortical synapses express postsynaptic kainate receptors as well as AMPA receptors; however, the two receptor subtypes do not colocalize. During the critical period for experience-dependent plasticity, the kainate-receptor contribution to transmission decreases; a similar decrease occurs when long-term potentiation is induced in vitro. This indicates that during development there is activity-dependent regulation of the expression of kainate receptors at thalamocortical synapses.
Asunto(s)
Corteza Cerebral/fisiología , Receptores de Ácido Kaínico/fisiología , Sinapsis/fisiología , Tálamo/fisiología , Animales , Corteza Cerebral/crecimiento & desarrollo , Potenciales Postsinápticos Excitadores , Técnicas In Vitro , Potenciación a Largo Plazo/fisiología , Ratas , Ratas Wistar , Receptores AMPA/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Tálamo/crecimiento & desarrolloRESUMEN
Although it plays a major inhibitory role in the adult mammalian CNS, gamma-aminobutyric acid (GABA) may have an excitatory function in developing neurons. The present study focuses on the dependence of glutamate on GABA to generate action potentials in developing hypothalamic neurons. Under conditions where glutamate by itself could not evoke an action potential, GABA facilitated glutamate-mediated depolarization to fire action potentials. This facilitation had a broad time window during the decaying phase of the GABA-mediated depolarization in developing neurons in culture. The glutamate-mediated depolarization was shunted only during the peak of GABA-mediated depolarization, but was facilitated after that. Similar results were obtained in the presence of 2-amino-5-phosphonopentanoic acid (AP5), indicating that GABA can facilitate glutamate responses independent of relief of the Mg2+ block of the N-methyl-D-aspartate (NMDA) receptor. This novel interaction between GABA- and glutamate-mediated excitation could play a role in strengthening neuronal circuits during early development and would exert a maximal effect if GABA and glutamate receptors were activated after a slight temporal delay.
Asunto(s)
Ácido Glutámico/farmacología , Hipotálamo/citología , Neuronas/efectos de los fármacos , Receptores AMPA/efectos de los fármacos , Receptores de Ácido Kaínico/efectos de los fármacos , Ácido gamma-Aminobutírico/fisiología , 2-Amino-5-fosfonovalerato/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Células Cultivadas , Antagonistas de Aminoácidos Excitadores/farmacología , Hipotálamo/embriología , Magnesio/farmacología , Neuronas/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Receptores AMPA/fisiología , Receptores de Ácido Kaínico/fisiología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidoresRESUMEN
Ionotropic glutamate receptors (iGluRs) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate subtype display lower permeability to Ca2+ than the N-methyl-D-aspartate (NMDA) subtype. The well-documented N/Q/R site on the M2 transmembrane segment (M2) is an important determinant of the distinct Ca2+ permeability exhibited by members of the non-NMDA receptor subfamily. This site, however, does not completely account for the different permeation properties displayed by non-NMDA and NMDA receptors, suggesting the involvement of other molecular determinants. We have identified additional molecular elements on M2 of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor GluR1 that specify its permeation properties. Higher permeability to divalent over monovalent cations is conferred on GluR1 by a tryptophan at position 577, whereas blockade by external divalent cations is imparted by an asparagine at position 582. Hence, the permeation properties of ionotropic glutamate receptors appear to be primarily specified by two distinct determinants on M2, the well-known N/Q/R site and the newly identified L/W site. These findings substantiate the notion that M2 is a structural component of the pore lining.
Asunto(s)
Calcio/metabolismo , Cationes Bivalentes/metabolismo , Permeabilidad de la Membrana Celular , Oocitos/fisiología , Receptores de Glutamato/química , Receptores de Glutamato/fisiología , Triptófano , Secuencia de Aminoácidos , Animales , ADN Complementario , Femenino , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Receptores AMPA/biosíntesis , Receptores AMPA/química , Receptores AMPA/fisiología , Receptores de Glutamato/biosíntesis , Receptores de Ácido Kaínico/biosíntesis , Receptores de Ácido Kaínico/química , Receptores de Ácido Kaínico/fisiología , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/fisiología , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , XenopusRESUMEN
The G-protein-coupled metabotropic glutamate receptor mGluR1 alpha and the ionotropic glutamate receptor GluR6 were examined for posttranslational palmitoylation. Recombinant receptors were expressed in baculovirus-infected insect cells or in human embryonic kidney cells and were metabolically labeled with [3H]palmitic acid. The metabotropic mGluR1 alpha receptor was not labeled whereas the GluR6 kainate receptor was labeled after incubation with [3H]palmitate. The [3H]palmitate labeling of GluR6 was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation at a cysteine residue via a thioester bond. Site-directed mutagenesis was used to demonstrate that palmitoylation of GluR6 occurs at two cysteine residues, C827 and C840, located in the carboxyl-terminal domain of the molecule. A comparison of the electrophysiological properties of the wild-type and unpalmitoylated mutant receptor (C827A, C840A) showed that the kainate-gated currents produced by the unpalmitoylated mutant receptor were indistinguishable from those of the wild-type GluR6. The unpalmitoylated mutant was a better substrate for protein kinase C than the wild-type GluR6 receptor. These data indicate that palmitoylation may not modulate kainate channel function directly but instead affect function indirectly by regulating the phosphorylation state of the receptor.
Asunto(s)
Ácidos Palmíticos/metabolismo , Procesamiento Proteico-Postraduccional , Receptores de Ácido Kaínico/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Alanina , Animales , Autorradiografía/métodos , Secuencia de Bases , Línea Celular , Cisteína , ADN Complementario , Embrión de Mamíferos , Embrión no Mamífero , Humanos , Riñón , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Sistemas de Lectura Abierta , Ácido Palmítico , Fosforilación , Mutación Puntual , Proteína Quinasa C/metabolismo , Receptores de Ácido Kaínico/biosíntesis , Receptores de Ácido Kaínico/fisiología , Receptores de Glutamato Metabotrópico/biosíntesis , Receptores de Glutamato Metabotrópico/fisiología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Spodoptera , Transfección , Tritio , Receptor de Ácido Kaínico GluK2RESUMEN
An essential part of the excitatory afferent input to the thalamus is mediated by glutamate receptors of the AMPA/kainate type. In contrast to other regions of the mammalian CNS, the biophysical properties of these receptors have not been investigated in thalamic neurones. Using a fast transmitter application system we studied L-glutamate activated currents of cultured neurones in the whole cell and outside-out patch configuration. Current-voltage relationships and dose-response curves of whole cell recordings were in close correspondence to results obtained from other brain areas. Analysis of outside-out patch currents revealed two types of desensitization time constants of 3.0 and 10.2 ms, with the former close to the time constant of decay of miniature glutamatergic synaptic currents.