Introduction 1 2 3 4 5 6 7 7 8 9 10 11 The aim of this study was to explore the effects of Aβ(25-35) treatment on main transporters implicated in sustaining synaptic release of amino acids, i.e. the glutamine transporters SN1 and SAT1 and the vesicular glutamate transporters VGLUT1 and VGLUT2, in two different in vitro models: neuronal cell cultures and mixed cell cultures of rat cerebral cortex. Experimental procedures Cell cultures 12 ○ g l 6 2 ○ Primary mixed cell cultures were prepared from the cerebral cortices of 1-day-old (P1) neonatal Wistar rats and plated on poly-L-lysine-treated coverslips (see above). The dissociated cortical cells were suspended in Neurobasal-A medium containing 2% B27 and 1% GlutaMAX I and maintained as described for E18 cells. Rats were kept according to nationally and internationally approved conditions. Culture media and reagents were from Gibco, Invitrogen Corporation (Carlsbad, CA, USA). Incubation with Aβ peptide Neuronal and mixed cell cultures were treated with Aβ(25-35) (Sigma-Aldrich, St.Louis, MO, USA) at final concentrations of 3 and 10 μM, for 12 or 24 h. In this study the B27-containing medium was removed from the cultures at day 6. Cells were washed twice with DMEM or Neurobasal-A medium and then incubated in medium for 12 and 24 h in the absence or presence of Aβ(25-35). Controls received no peptide. Antibodies 13 14 15 16 15 Immunocytochemical staining 3  Quantitative analysis of immunoreactivities 1 Fig. 1 a  c  e b  a d  e  c e f  c e f c  Data analysis 17 http://www.r-project.org/ Results Cell types 1 15 1 1 1 2 Fig. 2 a b c d a d  Survey of preparations double stained for MAP2 and either VGLUT1 or VGLUT2 indicated that a large proportion of the neurons were VGLUT immunoreactive. Similarly, double staining for MAP2 and SAT1 suggested that most of the neurons in the cultures contained SAT1. Effects of Aβ in mixed cell cultures 2 1 2 3 3 Fig. 3 2 a  c  e b  d  f c  d e  f n P P P  3 P P 3 P P P P Lack of effect of Aβ in neuronal cell cultures 4 5 5 5 P Fig. 4 a  b c  d a  c b  d  Fig. 5 Lack of effect of Aβ on SAT1 immunoreactivity in E18 cortical neuronal cells (cultured without glia). Data were obtained and presented as in Fig. 3  P P P P P Discussion [ 9 13 14 12 5 18 The effect of Aβ on neuronal SAT1 was observed in cultures containing neurons in close apposition to astroglial cells, but not in neuronal cultures without glia. This suggests that Aβ does not produce the effect by acting directly on the neurons, but via the glial cells, an idea gaining some support from the observed reduction in astroglial cell size. However, as the astrocytes had only slightly reduced size and normal levels of their marker protein SN1, the changes in neuronal SAT1 cannot be ascribed simply to toxic damage in the glia. 19 20 21 22 23 24 25 24 26 27 4 28