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

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

Andrew T. Kempsell

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