Modification of Gene Expression in Xenopus laeves Oocytes

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3.6.1. cRNA Synthesis and Oocyte Expression

Complementary RNAs (cRNAs) are synthesized from corresponding complementary DNAs (cDNAs) coding for the receptor gene of interest such as the glutamate receptor and are individually injected into Xenopus laeves oocytes individually or in desired receptor subunit combinations of 16-20 ng per subunit using a microinjector and prepared for electrophysiological recording.

3.6.2. Electrophysiological Recording

1. After detecting receptor expression, oocytes were placed in a 100-|L recording chamber perfused with modified Barth's solution and voltage-clamped using two microelectrodes at a holding potential of -70 mV.

2. Agonist-activated receptor currents were measured and recorded in response to known agonist concentrations such as 200 |M kainic acid used to measure AMPA receptor currents in oocytes.

3. Cannabinoids, endocannabinoids, alcohol, and other abused substances of interest are either co-applied or preapplied for a measured period of time such as 30-60 s with a period of about 5 min between applications to allow for drug washout and receptor recovery from desensitization. A schematic illustration of the expression system and electrophysiological recording is presented in Fig. 6.

Electrophysiology Oocyte

Fig. 6. Schematic illustration of receptors and ion channels in expressed Xenopus laeves oocytes for the study of the modification of receptors or ion channels by application of cannabinoids, endocannabinoids, or alcohol following current activation by 200 |M kainic acid in the two-microelectrode voltage-clamp setup.

Fig. 6. Schematic illustration of receptors and ion channels in expressed Xenopus laeves oocytes for the study of the modification of receptors or ion channels by application of cannabinoids, endocannabinoids, or alcohol following current activation by 200 |M kainic acid in the two-microelectrode voltage-clamp setup.

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