Assays for measuring extracellular GABA levels and cell migration rate in acute slices.
The postnatal subventricular zone (SVZ) contains the largest pool of dividing and migrating neural precursors in the adult rodent brain. Neuronal precursors migrate throughout the SVZ and along the rostral migratory stream (RMS) towards the olfactory bulb where they differentiate into interneurons. To facilitate the investigation of cell migration in the SVZ and RMS, an inexpensive migration assay was developed for use in acute brain slices. Acute sagittal slices were kept at 37 degrees C in 5% O2/95% CO2-saturated solution and migrating cells in the SVZ and RMS were visualized using an upright infrared-differential interference contrast microscope. Time-lapse movies were acquired to identify the direction and measure the speed of cell migration. The neurotransmitter GABA and inhibitors of GABA receptors or transporters can be bath applied to determine the function of endogenous GABA on the direction and speed of cell migration. In parallel, the levels of endogenous GABA released from acute SVZ or RMS explants were measured with mass spectrometry. Additional techniques such as electrophysiology and immunohistochemistry confirmed the identity of cells as neuronal precursors and characterized the expression of GABA receptors and transporters. This report describes how modulations in the direction and speed of neuronal precursor migration can be accurately monitored and how changes in local GABA levels can be measured. The described techniques can be used to identify the endogenous factors that regulate cell migration. Identifying such factors is essential for the future therapeutic use of SVZ cells to replace damaged or lost cells.
Bolteus
Anna J
AJ
Department of Neurosurgery, Yale University School of Medicine, New Haven, CT 06520-8082, USA.
Garganta
Cheryl
C
Bordey
Angélique
A
D000818:Animals; D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D002465:Cell Movement / Q000502:physiology*; D050485:GABA Plasma Membrane Transport Proteins; D007150:Immunohistochemistry / Q000379:methods; D020547:Lateral Ventricles / Q000166:cytology / Q000254:growth & development; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D018715:Microscopy, Video / Q000295:instrumentation / Q000379:methods; D009448:Neurochemistry / Q000295:instrumentation / Q000379:methods; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D009924:Organ Culture Techniques / Q000295:instrumentation / Q000379:methods; D018079:Receptors, GABA / Q000187:drug effects / Q000378:metabolism; D013234:Stem Cells / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D013687:Telencephalon / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology
D050485:GABA Plasma Membrane Transport Proteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D018079:Receptors, GABA; D005680:gamma-Aminobutyric Acid