isolation-sensory-neurons-aplysia-californica-for-patch-clamp

Isolation of Sensory Neurons of <em>Aplysia californica</em> for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

University of Miami

D-Aspartate (D-Asp) can substitute for L-glutamate (L-Glu) at excitatory Glu receptors, and occurs as free D-Asp in the mammalian brain. D-Asp electrophysiological responses were studied as a potential correlate of aging in the California sea hare, Aplysia californica. Whole cell voltage- and current clamp measurements were made from primary neuron cultures of the pleural ganglion (PVC) and buccal ganglion S cluster (BSC) in 3 egg cohorts at sexual maturity and senescence. D-Asp activated an inward current at the hyperpolarized voltage of -70 mV, where molluscan NMDA receptors open free of constitutive block by Mg(2+). Half of the cells responded to both D-Asp and L-Glu while the remainder responded only to D-Asp or L-Glu, suggesting that D-Asp activated non-Glu channels in a subpopulation of these cells. The frequency of D-Asp-induced currents and their density were significantly decreased in senescent PVC cells but not in senescent BSC cells. These changes in sensory neurons of the tail predict functional deficits that may contribute to an overall decline in reflexive movement in aged Aplysia.

Changes in D-aspartate ion currents in the Aplysia nervous system with aging.

D-Aspartate (D-Asp) peut remplacer L-glutamate (Glu-L) au niveau des r&eacute;cepteurs excitateurs Glu et se produit comme gratuit D-Asp dans le cerveau des mammif&egrave;res. D-Asp r&eacute;ponses &eacute;lectrophysiologiques ont &eacute;t&eacute; &eacute;tudi&eacute;s comme indicateur potentiel de vieillissement chez le li&egrave;vre de mer de Californie, Aplysia californica. Tension de cellules enti&egrave;res - et mesures de pince de courant ont &eacute;t&eacute; faites de cultures primaires de neurones du ganglion pleurale (PVC) et cluster de ganglion vestibulaire S (BSC) en 3 cohortes d&#39;oeuf &agrave; la s&eacute;nescence et la maturit&eacute; sexuelle. D-Asp activ&eacute; un courant entrant &agrave; la tension hyperpolaris&eacute;e de -70 mV, o&ugrave; les r&eacute;cepteurs NMDA mollusques ouverts sans bloc constitutif de Mg(2+). La moiti&eacute; des cellules a r&eacute;pondu &agrave; la fois D-Asp et Glu-L tandis que le reste a r&eacute;pondu qu&#39;aux D-Asp ou L-Glu, ce qui laisse supposer que le D-Asp activ&eacute; canaux non-Glu dans une sous-population de ces cellules. La fr&eacute;quence des courants induits par D-Asp et leur densit&eacute; ont consid&eacute;rablement diminu&eacute; dans les cellules s&eacute;nescentes de PVC mais pas dans les cellules s&eacute;nescentes de BSC. Ces changements dans les neurones sensoriels de la queue pr&eacute;disent des d&eacute;ficits fonctionnels qui peuvent contribuer &agrave; une diminution globale de mouvement r&eacute;flexif de l&#39;aplysie &acirc;g&eacute;.

Changements dans les courants ioniques D-aspartate dans le syst&egrave;me nerveux Aplysia avec le vieillissement.

D-aspartato (D-Asp) pu&ograve; sostituire L-glutammato (L-Glu) presso eccitatori Glu recettori e si verifica come libero D-Asp nel cervello dei mammiferi. D-Asp risposte elettrofisiologiche sono state studiate come un correlato potenziale di invecchiamento nella lepre di mare California, Aplysia californica. Cella intera tensione - e le misure di corrente morsetto sono state fatte da culture primarie neurone del ganglio pleurico (PVC) e cluster di ganglio vestibolare S (BSC) in 3 coorti di uovo a senescenza e maturit&agrave; sessuale. D-Asp attivato un perfezionamento attivo corrente alla tensione hyperpolarized di -70 mV, dove cotti i recettori NMDA aperti privo di blocco costitutivo di Mg(2+). Met&agrave; delle cellule ha risposto sia D-Asp e L-Glu, mentre il resto ha risposto solo a D-Asp o L-Glu, suggerendo che il D-Asp attivato canali non-Glu in una sottopopolazione di queste cellule. La frequenza delle correnti D-Asp-indotta e la loro densit&agrave; erano significativamente diminuiti in celle senescenti in PVC, ma non in celle senescenti BSC. Questi cambiamenti nei neuroni sensoriali della coda prevedono deficit funzionali che possono contribuire a un generale declino nel movimento riflessivo in Aplysia invecchiato.

Cambiamenti nelle correnti dello ione D-aspartato Aplysia del sistema nervoso con l&#39;invecchiamento.

Le composant de r&eacute;cepteurs non-L-glutamate (Glu-L) des courants de D-aspartate (D-Asp) dans Aplysia californica neurones du cluster (BSC) S buccales a &eacute;t&eacute; &eacute;tudi&eacute;e avec bride de tension de cellules enti&egrave;res pour diff&eacute;rencier des r&eacute;cepteurs activ&eacute;s par les autres agonistes de la bien connues du syst&egrave;me nerveux Aplysia et &eacute;tudier les m&eacute;canismes modulateurs des courants de D-Asp associ&eacute;s &agrave; la plasticit&eacute; synaptique. L&#39;ac&eacute;tylcholine (ACh) et courants excitateurs de la s&eacute;rotonine (5-HT) activ&eacute;s &agrave; germes entiers avec des relations de tension actuelle similaires &agrave; D-Asp. Ces courants, cependant, ont &eacute;t&eacute; pharmacologiquement distincts de D-Asp. ACh courants ont &eacute;t&eacute; bloqu&eacute;s par hexam&eacute;thonium (C6) et tubocurarine (D-TC), tandis que les courants D-Asp ne sont pas affect&eacute;s. 5-HT courants ont &eacute;t&eacute; bloqu&eacute;s par granis&eacute;tron et le m&eacute;thysergide (MES), tandis que les courants D-Asp ne sont pas affect&eacute;s. &Agrave; l&#39;inverse, m&ecirc;me si (acid(PPDA) 2S,3R)-1-(Phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic bloqu&eacute; courants D-Asp, il n&#39;avait aucun effet sur les ACh ou courants de la 5-HT. Comparaison de la zone de charge d&eacute;crite par les courants induits par l&#39;ACh ou 5-HT ind&eacute;pendamment de, ou avec, D-Asp sugg&egrave;re d&#39;activation des r&eacute;cepteurs distincts par les 3 agonistes. Frais comparaison avec L-Glu, cependant, a sugg&eacute;r&eacute; certains chevauchements entre les r&eacute;cepteurs de la L-Glu et D-Asp. Exposition de dix minutes &agrave; 5-HT facilitation induite des r&eacute;ponses D-Asp-&eacute;voqu&eacute; dans les neurones de la BSC. Cet effet a &eacute;t&eacute; imit&eacute; par ester de phorbol, sugg&eacute;rant que la prot&eacute;ine kinase C (PKC) a &eacute;t&eacute; impliqu&eacute;.

Les r&eacute;cepteurs ionotropiques unique pour D-aspartate sont une cible pour la plasticit&eacute; synaptique induite par la s&eacute;rotonine dans Aplysia californica.

The non-L-glutamate (L-Glu) receptor component of D-aspartate (D-Asp) currents in Aplysia californica buccal S cluster (BSC) neurons was studied with whole cell voltage clamp to differentiate it from receptors activated by other well-known agonists of the Aplysia nervous system and investigate modulatory mechanisms of D-Asp currents associated with synaptic plasticity. Acetylcholine (ACh) and serotonin (5-HT) activated whole cell excitatory currents with similar current voltage relationships to D-Asp. These currents, however, were pharmacologically distinct from D-Asp. ACh currents were blocked by hexamethonium (C6) and tubocurarine (D-TC), while D-Asp currents were unaffected. 5-HT currents were blocked by granisetron and methysergide (MES), while D-Asp currents were unaffected. Conversely, while (2S,3R)-1-(Phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic acid(PPDA) blocked D-Asp currents, it had no effect on ACh or 5-HT currents. Comparison of the charge area described by currents induced by ACh or 5-HT separately from, or with, D-Asp suggests activation of distinct receptors by all 3 agonists. Charge area comparisons with L-Glu, however, suggested some overlap between L-Glu and D-Asp receptors. Ten minute exposure to 5-HT induced facilitation of D-Asp-evoked responses in BSC neurons. This effect was mimicked by phorbol ester, suggesting that protein kinase C (PKC) was involved.

Unique ionotropic receptors for D-aspartate are a target for serotonin-induced synaptic plasticity in Aplysia californica.

I-L-glutammato (L-Glu) componente del recettore delle correnti D-aspartato (D-Asp) in Aplysia californica buccali dei neuroni di cluster (BSC) S &egrave; stato studiato con morsetto tensione cella intera per differenziarlo dai recettori attivati da altri ben noti agonisti del sistema nervoso Aplysia e indagare meccanismi modulatrice delle correnti D-Asp associate con plasticit&agrave; sinaptica. Acetilcolina (ACh) e serotonina (5-HT) attivato intera cellula correnti eccitatorie con simili rapporti di tensione corrente di D-Asp. Queste correnti, tuttavia, erano farmacologicamente distinte da D-Asp. ACh correnti sono state bloccate da hexamethonium (C6) e tubocurarina (D-TC), mentre le correnti D-Asp erano inalterate. 5-HT correnti sono state bloccate da granisetron e metisergide (MES), mentre le correnti D-Asp sono state influenzate. Al contrario, mentre (2S,3R)-1-(Phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic acid(PPDA) bloccato le correnti D-Asp, ha avuto alcun effetto su ACh o correnti di 5-HT. Confronto tra la zona di carica descritta da correnti indotte dall&#39;ACh o separatamente da, o con 5-HT, D-Asp suggerisce l&#39;attivazione di recettori distinti da tutti gli 3 agonisti. Carica zona paragoni con L-Glu, tuttavia, ha suggerito qualche sovrapposizione tra recettori L-Glu e D-Asp. Esposizione di dieci minuti a 5-HT facilitazione indotta di D-Asp-evocato le risposte nei neuroni BSC. Questo effetto &egrave; stato imitato da estere del forbolo, suggerendo che protein chinasi C (PKC) &egrave; stato coinvolto.

I recettori ionotropici unica per D-aspartato sono un bersaglio per plasticit&agrave; sinaptica indotta da serotonina in Aplysia californica.

D-Aspartate (D-Asp) activates an excitatory current in neurons of Aplysia californica. Although D-Asp is presumed to activate a subset of L-glutamate (L-Glu) channels, the identities of putative d-Asp receptors and channels are unclear. Whole cell voltage- and current-clamp studies using primary cultures of Aplysia buccal S cluster (BSC) neurons were executed to characterize D-Asp-activated ion channels. Both D-Asp and L-Glu evoked currents with similar current-voltage relationships, amplitudes, and relatively slow time courses of activation and inactivation when agonists were pressure applied. D-Asp-induced currents, however, were faster and desensitized longer, requiring 40 s to return to full amplitude. Of cells exposed to both agonists, 25% had D-Asp- but not L-Glu-induced currents, suggesting a receptor for D-Asp that was independent of l-Glu receptors. D-Asp channels were permeable to Na(+) and K(+), but not CaÂ²âº, and were vulnerable to voltage-dependent MgÂ²âº block similarly to vertebrate NMDA receptor (NMDAR) channels. d-Asp may activate both NMDARs and non-l-Glu receptors in the nervous system of Aplysia.

Physiological evidence that D-aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica neurons.

D-Aspartate (D-Asp) active un courant excitateur dans les neurones d&#39;Aplysia californica. Bien que D-Asp est cens&eacute; activer un sous-ensemble de canaux de L-glutamate (Glu-L), les identit&eacute;s des canaux et des r&eacute;cepteurs putatifs de d-Asp ne sont pas claires. &Eacute;tudes de bride de tension et de courant de germes entiers &agrave; l&#39;aide de cultures primaires de Aplysia buccales S cluster (BSC) neurones ont &eacute;t&eacute; ex&eacute;cut&eacute;s afin de caract&eacute;riser les canaux ioniques activ&eacute;s par D-Asp. Les D-Asp et Glu-L courants avec des relations courant-tension similaires, amplitudes et relativement lentement de l&#39;activation et l&#39;inactivation des cours &agrave; temps lorsque les agonistes ont subi une pression. Induite par la D-Asp courants, cependant, &eacute;taient plus rapides et d&eacute;sensibilis&eacute;s plus longue, n&eacute;cessitant 40 s pour revenir en pleine amplitude. Des cellules expos&eacute;es aux deux agonistes, 25 % avaient D-Asp, mais pas L-Glu-induit courants, ce qui sugg&egrave;re un r&eacute;cepteur pour D-Asp qui &eacute;tait ind&eacute;pendant des r&eacute;cepteurs l-Glu. D-Asp canaux &eacute;tait perm&eacute;ables &agrave; Na et k, mais pas Ca&sup2;&#x207A; et vuln&eacute;rables au bloc Mg&sup2;&#x207A; voltage-d&eacute;pendants de la m&ecirc;me fa&ccedil;on aux canaux de r&eacute;cepteur (NMDAR) vert&eacute;br&eacute;s NMDA. d-Asp peut activer les r&eacute;cepteurs NMDA et non-l-Glu r&eacute;cepteurs dans le syst&egrave;me nerveux d&#39;Aplysia.

Donn&eacute;es physiologiques que D-aspartate active un courant distinct de courants de r&eacute;cepteur de glutamate ionotropiques dans les neurones Aplysia californica.

D-aspartato (D-Asp) si attiva una corrente eccitatoria in neuroni di Aplysia californica. Anche se si presume che il D-Asp attivare un sottoinsieme dei canali L-glutammato (L-Glu), le identit&agrave; di putativi recettori d-Asp e canali sono poco chiare. Gli studi del morsetto di tensione e di corrente cella intera utilizzando colture primarie di neuroni di Aplysia buccali S cluster (BSC) furono giustiziati per caratterizzare le scanalature dello ione D-Asp-attivato. D-Asp sia L-Glu evocato correnti con simili rapporti corrente-tensione, ampiezze e relativamente lento corsi a tempo di attivazione ed inattivazione quando agonisti erano pressione applicata. Correnti D-Asp-indotta, tuttavia, erano pi&ugrave; veloci e desensibilizzati pi&ugrave; lunghi, che richiedono 40 s per tornare alla piena ampiezza. Delle cellule esposte a entrambi agonisti, 25% aveva D-Asp-ma le correnti non L-Glu-indotta, suggerendo un recettore per D-Asp che era indipendente dei recettori l-Glu. D-Asp canali erano permeabile a Na(+) e K(+), ma non Ca&sup2;&#x207A; e vulnerabili al blocco Mg&sup2;&#x207A; voltaggio-dipendenti Analogamente ai vertebrati canali NMDA receptor (NMDAR). d-Asp pu&ograve; attivare sia NMDARs e -l-Glu recettori nel sistema nervoso di Aplysia.

Prove fisiologiche che D-aspartato attiva una corrente distinta da ionotropici le correnti di recettore del glutammato in Aplysia californica neuroni.

D-Aspartate (D-Asp) activates a nonspecific cation current of unknown identity independent of L-glutamate (L-Glu) in neurons of Aplysia californica. Whole-cell voltage clamp studies were conducted using primary cultures of Aplysia buccal S cluster (BSC) neurons to characterize these receptor channels pharmacologically. The N-methyl-D-aspartate receptor (NMDAR) coagonist glycine potentiated D-Asp currents only at -30 mV, while D-serine did not potentiate D-Asp currents at any amplitude. Portions of D-Asp currents were blocked by the L-Glu antagonists kynurenate, DL-2-amino-5-phosphonopentanoic acid (APV), (2S,3R)-1-(phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA), and 1,3-dihydro-5-[3-[4-(phenylmethyl)-1-2H-benzimidazol-2-one (TCS46b), suggesting that L-Glu channels, particularly NMDAR-like channels, may partially contribute to D-Asp whole-cell currents. In contrast, L-Glu currents were unaffected by APV, and showed greater block by kynurenate, suggesting that D-Asp and L-Glu act, in part, at different sites. The excitatory amino acid transport blocker DL-threo-b-Benzyloxyaspartic acid (TBOA) blocked a fraction of D-Asp currents, suggesting that currents associated with these transporters also contribute. Non-NMDA L-GluR antagonists that preferentially block alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate receptors significantly increased D-Asp currents, suggesting a possible allosteric potentiating effect of these antagonists on D-Asp receptors. L-Glu-induced currents were significantly reduced in the presence of bath-applied D-Asp, whereas bath-applied L-Glu had no effect on D-Asp-induced currents. The mixed effects of these agents on D-Asp-induced currents in Aplysia illustrate that the underlying channels are not uniformly characteristic of any known agonist associated channel type.

Pharmacological evidence that D-aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica.

D-Aspartate (D-Asp) active un courant cationique non sp&eacute;cifique d&#39;identit&eacute; inconnue ind&eacute;pendante de L-glutamate (Glu-L) dans les neurones d&#39;Aplysia californica. Cellule enti&egrave;re pince &eacute;tudes ont &eacute;t&eacute; men&eacute;es &agrave; l&#39;aide de cultures primaires de Aplysia buccales S neurones de cluster (BSC) pour caract&eacute;riser ces canaux r&eacute;cepteur pharmacologiquement. La glycine de coagonist N-m&eacute;thyl-D-aspartate receptor (NMDAR) potentialise les courants D-Asp seulement &agrave; -30 mV, tandis que la D-s&eacute;rine n&#39;a pas potentialis&eacute; courants D-Asp &agrave; n&#39;importe quel amplitude. Portions de courants D-Asp ont &eacute;t&eacute; bloqu&eacute;es par la L-Glu antagonistes kynur&eacute;nate, acide DL-2-amino-5-phosphonopentano&iuml;que (APV), (l&#39;acide 2S,3R)-1-(phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic (PPDA) et 1,3-dihydro-5-[3-[4-(phenylmethyl)-1-2H-benzimidazol-2-one (TCS46b), ce qui sugg&egrave;re que le L-Glu canaux, particuli&egrave;rement NMDAR-comme canaux, peuvent contribuer partiellement &agrave; des courants de germes entiers D-Asp. En revanche, L-Glu courants ne sont pas affect&eacute;s par l&#39;APV et a montr&eacute; le grand bloc de kynur&eacute;nate, ce qui sugg&egrave;re que D-Asp et Glu-L agissent, en partie, sur des sites diff&eacute;rents. L&#39;acide de bloqueur DL-thr&eacute;o-b-benzoyloxyaspartique de transport des acides amin&eacute;s excitateurs (TBOA) bloqu&eacute; une fraction des courants de D-Asp, sugg&eacute;rant que les courants associ&eacute;s &agrave; ces transporteurs contribuent &eacute;galement. Non-NMDA L-GluR antagonistes qui bloquent pr&eacute;f&eacute;rentiellement en acide alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic (AMPA) / r&eacute;cepteurs ka&iuml;nate a consid&eacute;rablement augment&eacute; les courants D-Asp, sugg&eacute;rant un possible effet potentialisateur allost&eacute;rique de ces antagonistes sur les r&eacute;cepteurs D-Asp. Courants induits par L-Glu ont chut&eacute; en pr&eacute;sence de bain-appliqu&eacute; D-Asp, tandis que le bain-applique L-Glu n&#39;avait aucun effet sur les courants induits par D-Asp. Les effets mixtes de ces agents sur les courants D-Asp-induite de l&#39;aplysie illustrent que les canaux sous-jacents ne sont pas uniform&eacute;ment caract&eacute;ristiques de n&#39;importe quel type de canal connu agoniste associ&eacute;.

Preuve pharmacologique que D-aspartate active un courant distinct de courants de r&eacute;cepteur de glutamate ionotropiques dans Aplysia californica.

D-aspartato (D-Asp) si attiva una corrente cationico aspecifico di sconosciuta identit&agrave; indipendente del L-glutammato (L-Glu) nei neuroni di Aplysia californica. Cellule intere tensione pinza studi sono stati condotti utilizzando colture primarie di neuroni di Aplysia buccali S cluster (BSC) per caratterizzare questi canali recettore farmacologicamente. La glicina N-metil-D-aspartato recettore (NMDAR) coagonist potenziate le correnti D-Asp solo a -30 mV, mentre D-serina ha fatto non potenziare le correnti D-Asp presso qualsiasi ampiezza. Porzioni di correnti di D-Asp sono stati bloccati dalla L-Glu antagonisti chinurenato, acido DL-2-ammino-5-phosphonopentanoic (APV), (acido 2S,3R)-1-(phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic (PPDA) e 1,3-dihydro-5-[3-[4-(phenylmethyl)-1-2H-benzimidazol-2-one (TCS46b), suggerendo che L-Glu, canali, particolarmente NMDAR-come canali, parzialmente pu&ograve; contribuire alle correnti di cellule intere di D-Asp. Al contrario, L-Glu correnti sono state influenzate da APV e ha mostrato una maggiore blocco da chinurenato, suggerendo che D-Asp e L-Glu agiscono, in parte, a diversi siti. L&#39;acido di stampo DL-Treo-b-Benzyloxyaspartic trasporto degli aminoacidi eccitatori (TBOA) bloccato una frazione delle correnti D-Asp, suggerendo che le correnti associate con questi trasportatori anche contribuiscano. L-GluR non-NMDA antagonisti che preferenzialmente bloccano l&#39;acido alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic (AMPA) / recettori Kainato aumentato significativamente le correnti D-Asp, suggerendo un possibile effetto allosterico cumulo di questi antagonisti sui recettori D-Asp. L-Glu-indotta delle correnti si sono sensibilmente ridotti in presenza di vasca applicato D-Asp, mentre applicato a bagno L-Glu ha avuto alcun effetto sulle correnti di D-Asp-indotta. Gli effetti misti di questi agenti sulle correnti di D-Asp-indotta in Aplysia illustrano che i canali sottostanti non sono uniformemente caratteristici di qualsiasi tipo di canale noto agonista associato.

Prove farmacologiche che D-aspartato attiva una corrente distinta da ionotropici le correnti di recettore del glutammato in Aplysia californica.

Ethanol exposure produces alterations in GABAergic signaling that are associated with dependence and withdrawal. Previously, we demonstrated that ethanol-induced protein kinase C (PKC) Î³ signaling selectively contributes to changes in GABAA Î±1 synaptic receptor activity and surface expression. Here, we demonstrate that protein kinase A (PKA) exerts opposing effects on GABAA receptor adaptations during brief ethanol exposure. Cerebral cortical neurons from day 0-1 rat pups were tested after 18 days in culture. Receptor trafficking was assessed by Western blot analysis, and functional changes were measured using whole-cell patch-clamp recordings of evoked and miniature inhibitory postsynaptic current (mIPSC) responses. One-hour ethanol exposure increased membrane-associated PKC and PKA, but steady-state GABAA Î±1 subunit levels were maintained. Activation of PKA by Sp-adenosine 3',5'-cyclic monophosphothioate triethylamine alone increased GABAA Î±1 subunit surface expression and zolpidem potentiation of GABA responses, whereas coexposure of ethanol with the PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine decreased Î±1 subunit expression and zolpidem responses. Exposure to the PKC inhibitor calphostin-C with ethanol mimicked the effect of direct PKA activation. The effects of PKA modulation on mIPSC decay Ï„ were consistent with its effects on GABA currents evoked in the presence of zolpidem. Overall, the results suggest that PKA acts in opposition to PKC on Î±1-containing GABAA receptors, mediating the GABAergic effects of ethanol exposure, and may provide an important target for the treatment of alcohol dependence/withdrawal.

Ethanol activation of protein kinase A regulates GABAA Î±1 receptor function and trafficking in cultured cerebral cortical neurons.

Exposition de l&#39;&eacute;thanol produit des alt&eacute;rations GABAergiques qui sont associ&eacute;s &agrave; la d&eacute;pendance et retrait de signalisation. Auparavant, nous avons d&eacute;montr&eacute; qu&#39;induite par l&#39;&eacute;thanol prot&eacute;ine kinase C (PKC) &gamma; signalisation s&eacute;lectivement contribue &agrave; des changements dans l&#39;activit&eacute; synaptique r&eacute;cepteur de GABAA &alpha;1 et model&eacute;. Ici, nous d&eacute;montrons que la prot&eacute;ine kinase A (PKA) exerce des effets adverses sur les adaptations du r&eacute;cepteur GABAA pendant l&#39;exposition br&egrave;ve de l&#39;&eacute;thanol. Neurones du cortex c&eacute;r&eacute;bral de ratons jour 0-1 ont &eacute;t&eacute; test&eacute;s apr&egrave;s 18 jours de culture. R&eacute;cepteur de trafic a &eacute;t&eacute; &eacute;valu&eacute; par analyse par Western blot et changements fonctionnels ont &eacute;t&eacute; mesur&eacute;s en utilisant des enregistrements de patch-clamp de la cellule enti&egrave;re des r&eacute;ponses &eacute;voqu&eacute;es et miniature courant postsynaptique inhibiteur (mIPSC). Exposition d&#39;une heure l&#39;&eacute;thanol augmente associ&eacute;es aux membranes PKC et PKA, mais &eacute;quilibre GABAA &alpha;1 sous-unit&eacute; niveaux ont &eacute;t&eacute; maintenus. Activation de PKA par Sp-ad&eacute;nosine 3&#39;, 5&#39;-cyclique monophosphothioate tri&eacute;thylamine seul augment&eacute; de GABAA &alpha;1 sous-unit&eacute; surface expression et zolpidem potentialisation des r&eacute;ponses de GABA, tandis que coexposure de l&#39;&eacute;thanol avec l&#39;inhibiteur PKA-Rp-ad&eacute;nosine 3&#39;, 5&#39;-cyclique monophosphothioate tri&eacute;thylamine diminue l&#39;expression de sous-unit&eacute; &alpha;1 et zolpidem r&eacute;ponses. Exposition &agrave; l&#39;inhibiteur PKC calphostine-C avec de l&#39;&eacute;thanol a reproduit l&#39;effet de l&#39;activation directe de PKA. Les effets de modulation de la PKA sur mIPSC carie &tau; correspondaient &agrave; ses effets sur les courants de GABA &eacute;voqu&eacute;es en pr&eacute;sence de zolpidem. Dans l&#39;ensemble, les r&eacute;sultats sugg&egrave;rent que le PKA agit &agrave; l&#39;encontre de PKC sur &alpha;1-contenant des r&eacute;cepteurs GABAA, m&eacute;diation des effets GABAergiques d&#39;exposition de l&#39;&eacute;thanol et peut fournir une cible importante pour le traitement de la d&eacute;pendance/sevrage de l&#39;alcool.

Activation de l&#39;&eacute;thanol de la prot&eacute;ine kinase A r&eacute;gule la fonction des r&eacute;cepteurs GABAA &alpha;1 et traite de culture c&eacute;r&eacute;brale des neurones corticaux.

Esposizione di etanolo produce alterazioni GABAergico segnalazione che sono associati a dipendenza e astinenza. In precedenza, abbiamo dimostrato che etanolo-indotta della proteina chinasi C (PKC) &gamma; segnalazione selettivamente contribuisce ai cambiamenti in attivit&agrave; sinaptica del ricevitore di GABAA &alpha;1 e l&#39;espressione di superficie. Qui, noi dimostrare che tale proteina chinasi A (PKA) esercita effetti opposti su adattamenti del recettore GABAA durante l&#39;esposizione breve etanolo. Neuroni corticali cerebrali da cuccioli di ratto giorno 0-1 sono stati testati dopo 18 giorni in coltura. Traffico di recettore &egrave; stata valutata mediante analisi Western blot, e modifiche funzionali sono state misurate utilizzando registrazioni di cellule intere patch-clamp di risposte corrente postsynaptic inibitorio evocato e in miniatura (mIPSC). L&#39;esposizione ora etanolo aumentato associato membrana PKC e PKA, ma stazionario GABAA &alpha;1 unit&agrave; secondaria livelli sono stati mantenuti. Attivazione della PKA di Sp-adenosina 3&#39;, 5&#39;-ciclico monophosphothioate trietilammina solo aumentata GABAA &alpha;1 unit&agrave; secondaria superficie espressione e zolpidem potenziamento delle risposte di GABA, mentre coexposure di etanolo con l&#39;inibitore PKA Rp-adenosina 3&#39;, 5&#39;-ciclico monophosphothioate trietilammina diminuita espressione di subunit&agrave; &alpha;1 e risposte di zolpidem. L&#39;esposizione al PKC inibitore calphostin-C con etanolo imitato l&#39;effetto di attivazione diretta di PKA. Gli effetti di modulazione PKA su mIPSC decadimento &tau; erano coerenti con i suoi effetti sulle correnti GABA evocate in presenza di zolpidem. Nel complesso, i risultati suggeriscono che la PKA agisce in opposizione PKC sui recettori GABAA &alpha;1-contenente, mediando gli effetti di GABAergic di esposizione di etanolo e pu&ograve; fornire un obiettivo importante per il trattamento della dipendenza/astinenza da alcol.

Stephen L. Carlson

Division of Marine Biology and Fisheries,
Rosenstiel School of Marine and Atmospheric Sciences,
Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Sciences

University of Miami

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Stephen L. Carlson

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Marine Biology and Fisheries,Rosenstiel School of Marine and Atmospheric Sciences,Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Sciences

University of Miami

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Division of Marine Biology and Fisheries,
Rosenstiel School of Marine and Atmospheric Sciences,
Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Sciences