NA-DEPENDENT, FLUOXETINE-SENSITIVE SEROTONIN UPTAKE BY ASTROCYTES TISSUE-PRINTED FROM RAT CEREBRAL-CORTEX()

Previous studies have established that rat primary astrocyte cultures prepared from several brain regions of 1-4-d-old rats exhibit high-aff inity, Na+-dependent and fluoxetine-sensitive serotonin (5-HT) uptake with a K-m for 5-HT of 0.4 mu M and a K-i for fluoxetine of 23 nM, whi ch correspond to the characteristics for this transport for other brai n preparations. However, it is not known whether astrocytes in situ sh ow such uptake. We addressed this question by performing H-3- 5-HT upt ake experiments on cortical astrocytes, within 4 hr of isolating them from 6- and 21-d-old rats by the tissue-print technique. Quantitative autoradiography was combined with GFAP and neurofilament (NF) immunocy tochemistry to distinguish astrocytic from neuronal H-3-5-HT uptake. I n composition, the tissue-printed (TP) cells and processes were 60-70% GFAP(+) and 10-15% NF(+). H-3-5-HT uptake (0.3 mu M 5-HT, 3.4 mu Ci/m l) in both tissue-printed GFAP(+) astrocytes and NF(+) structures was sensitive to 1 PM fluoxetine and was also Na+ dependent. More than 90% of TP astrocytes from 6- and 21-d-old rats and 100% of NF(+) structur es from 21-d-old rats showed positive 3H-5-HT uptake (defined as great er than or equal to 31 grains/10(3) mu m(2)). The highest level of upt ake (greater than or equal to 191 grains/10(3) mu m(2)) was never obse rved in TP astrocytes but was exhibited by about half of the NF(+) str uctures. In other experiments we found that H-3-5-HT uptake by 6-d-old TP astrocytes was comparable to uptake by postnatal age-matched prima ry cultured astrocytes that were grown in fetal bovine serum (FBS). Ho wever, primary cultured astrocytes grown in horse serum showed lower u ptake than that observed with FBS, a finding similar to previous resul ts in cultures where H-3-5-HT uptake was measured per milligram of cel l protein. These results imply that high-affinity, Na+-dependent and f luoxetine-sensitive 5-HT uptake occurs in rat cortical astrocytes in s itu.