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) 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.

Andrew T. Kempsell

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