Na(+)-dependent, fluoxetine-sensitive serotonin uptake by astrocytes tissue-printed from rat cerebral cortex

Abstract

Previous studies have established that rat primary astrocyte cultures prepared from several brain regions of 1-4-d-old rats exhibit high-affinity, Na(+)-dependent and fluoxetine-sensitive serotonin (5-HT) uptake with a Km for 5-HT of 0.4 microM and a Ki for fluoxetine of 23 nM, which correspond to the characteristics for this transport for other brain preparations. However, it is not known whether astrocytes in situ show such uptake. We addressed this question by performing 3H-5-HT uptake 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) immunocytochemistry to distinguish astrocytic from neuronal 3H-5-HT uptake. In composition, the tissue-printed (TP) cells and processes were 60-70% GFAP (+) and 10-15% NF(+). 3H-5-HT uptake (0.3 microM 5-HT, 3.4 microCi/ml) in both tissue-printed GFAP(+) astrocytes and NF(+) structures was sensitive to 1 microM fluoxetine and was also Na+ dependent. More than 90% of TP astrocytes from 6- and 21-d-old rats and 100% of NF(+) structures from 21-d-old rats showed positive 3H-5-HT uptake (defined as > or = 31 grains/10(3) microns2). The highest level of uptake (> or = 191 grains/10(3) microns2) was never observed in TP astrocytes but was exhibited by about half of the NF(+) structures. In other experiments were found that 3H-5-HT uptake by 6-d-old TP astrocytes was comparable to uptake by postnatal age-matched primary cultured astrocytes that were grown in fetal bovine serum (FBS). However, primary cultured astrocytes grown in horse serum showed lower uptake than that observed with FBS, a finding similar to previous results in cultures where 3H-5-HT uptake was measured per milligram of cell protein. These results imply that high-affinity, Na(+)-dependent and fluoxetine-sensitive 5-HT uptake occurs in rat cortical astrocytes in situ.

Freshly isolated astrocyte (FIA) preparations: A useful single cell system for studying astrocyte properties

“…Abbreviations: FIA, freshly isolated astrocytes; K A , transient outward K current; K D , delayed outward rectifier; K IR , inward ("anomalous") rectifier; K OHM , passive or leak K ϩ current; K SI , slow inactivating inward K ϩ current; GLT-1, GLAST, glutamate/aspartate transporters (both are astrocytic); NE, norepinephrine; mGluR, metabotropic glutamate receptor; KA, kainate; SOD, superoxide dismutase; D1, dopamine D1 receptor. b References; (1) Swanson et al, 1997; (2) Rothstein et al, 1994; (3) Katz and Kimelberg, 1985; (4) Semenoff and Kimelberg, 1985; (5) Dave and Kimelberg, 1994;(6) Pickel and Chan, 1999; (7) Bowman and Kimelberg, 1984;(8) Kimelberg et al, 1997;(9) Backus et al, 1989; (10) Kimelberg, 1988; (11) Kettenmann and Schachner, 1985;(12) Garcia-Barcina and Matute, 1996; (13) Shelton and McCarthy, 1999;(14) Conti et al, 1996;(15) Rodriguez et al, 1999;(16) Porter and McCarthy, 1996;(17) Sontheimer, 1994;(18) Rutledge and Kimelberg, 1996;(19) Araque et al, 2000;(20) Bignami et al, 1980;(21) Norenberg, 1979;(22) Cammer and Tansey, 1988; (23) Raivich et al, 1999Raivich et al, . 2000.…”

The Pro-inflammatory Cytokine TNF-α Regulates the Activity and Expression of the Serotonin Transporter (SERT) in Astrocytes

“…However, in recent years, several reports have been published providing compelling evidence for the expression of functionally active SERT in glial cells lines as well as in primary astrocytes [10,[29][30][31]. High-affinity uptake of 5-HT by astrocytes is sensitive to inhibition by antidepressants such as imipramine and SSRIs, with a high degree of correlation noted between the Ki values for inhibition of 5-HT uptake by antidepressants in astrocytes and neurons, thus suggesting that the pharmacologically indistinguishable, highaffinity 5-HT uptake is mediated by SERT in both, glial and neuronal cells [30,32,33]. Furthermore, in recent years glial-derived SERT has been successfully visualized in rat brain tissue [34,35].…”

Long-term modulation of presynaptic 5-HT-output: Experimentally induced changes in cortical 5-HT-transporter density, tryptophan hydroxylase content and 5-HT innervation density

“…Fifth, the rate of 5-HT degradation may change under certain circumstances, e.g. by an increased production of tribulin, an endogenous MAO-inhibitor (Bhattacharya et al, 1988) or by increased or decreased uptake and metabolization of released 5-HT by astrocytes (Hertz, 1989;Dave and Kimelberg, 1994). Finally, even the density of 5-HT innervation and of 5-HT presynapses in terminal fields of 5-HT projections may decrease (due to degeneration and retraction of 5-HT nerve endings) or increase (due to collateral sprouting and formation of new synapses).…”