An in vitro model for studying the effects of continuous ethanol exposure on N-methyl-d-aspartate receptor function

Staurosporine decreases the intracellular distribution of mitochondria in astrocytes cocultured with cerebellar granule cells (CGCs). Micrograph of astrocytes from an 8 days in vitro [DIV] coculture with CGCs in which nuclei are identified with 4′,6′-diamino-2-phenylindole and mitochondria with MitoTracker Red CMXRos. Note the abundance of mitochondria and the distribution throughout the entire astrocytic cell body in control cells (A). However, mitochondrial distribution is attenuated in astrocytes that were treated overnight with 125 nM staurosporine (B). Post hoc analysis indicated that percent thresholded area generated by MitoTracker CMXRos was significantly less: 1116.8 ± 113.3 for staurosporine-treated cells compared with 2091.2 ± 335.6 for control astrocytes for ring one; t = 3.8, P ≤ .001. Similar results were observed for ring five in that an overnight 125 nM staurosporine treatment decreased CMXRos-generated pixel area to 718.0 ± 149 from control values of 1780.0 ± 386; t = 3.0, P ≤ .01. As stated in the Materials and Methods, nuclei from astrocytes were larger in diameter and were not as dense as CGC nuclei and thus were easily identified. CGC nuclei contained in these images are identified by arrows; scale bars = 20 μm.

Continuous ethanol exposure (CEE) does not alter the mitochondrial intracellular distribution in old or young days in vitro [DIV] cerebellar granule cell (CGC) astrocytic cocultures. Images were taken with a confocal microscope of CGCs that were five DIV at the start of CEE treatment (young). Mitochondria were distributed throughout the astrocytic cell body in control cells (A) as indicated by MitoTracker Red CMXRos-generated signal and this did not differ in astrocytes at (B) Day 5 or at (C) 72 + W. CGCs are indicated with small arrows and the single fibroblast by a large arrow. Images for similar experiments in older (21–22 DIV) at the start of CEE (old) indicate that intracellular mitochondrial distribution did not differ among (D) control, (E) Day 5, or (F) 72 + W time points. Images for the older DIV cocultures were taken with a Photometrics CoolSnap HQ cooled digital camera as described in the Materials and Methods section. The images for the older DIV CGCs were not acquired with a confocal microscope and only reflect one plane, a plane that is below the CGCs. Thus, there are no CGCs visible in images D–F. Summarized data for young and old DIV cocultures are shown in graphs G and H, respectively, as percent change from control values; scale bars = 20 μm.

Altered morphology of astrocytic cytoskeleton after treatment with the depolymerizing agent latrunculin. (A) Astrocytic cerebellar granule cell (CGC) cocultures (12 days in vitro [DIV]) were treated with 5 μM latrunculin A overnight, then fixed and stained with GFAP/Cy3. The percent thresholded area resultant of the GFAP/Cy3 staining is much greater in (A) control astrocytes compared with (B) the latrunculin A-treated cells in which a depolymerization of the F-actin cytoskeleton resulted in a complete breakdown of astrocytic morphology. Data were generated from five different fields of view from each coverslip taken from two control dishes and three latrunculin A-treated dishes; scale bar at 20 μm.

Continuous ethanol exposure (CEE) does not alter the integrity of the cytoskeleton of astrocytes contained in young or old cerebellar granule cell (CGC) cultures. Cocultures of CGCs and astrocytes were treated with 100 mM ethanol as described in the Materials and Methods section. Cells were then processed for identification of the cytoskeleton with GFAP/Cy3 at four different times post-CEE and micrographs were acquired with a confocal microscope. Note the lack of change in the morphology of the astrocytic cytoskeleton for (A) control, (B) Day 5, and at (C) 72 + W for young days in vitro [DIV] cocultures of astrocytes and CGCs. Representative images for CEE started at 21 + DIV cultures of CGCs and astrocytes are shown for (D) control, (E) Day 5, and (F) 72 + W also depict no change in astrocytic morphology attributed to CEE. Summarized data for young and old DIV cocultures are shown in graphs G and H, respectively, as percent change from control values; scale bar at 20 μm.

Decreases in GFAP/Cy3 thresholded signal are due to a decrease in the intensity and not due to a change in astrocytic morphology. Micrographs from a control (A) and a continuous ethanol exposure (CEE) dish of cocultured cerebellar granule cells (CGCs) and astrocytes (26 days in vitro [DIV]) Day 5 time point (B) taken from the same culture batch. Note that the shape of the CEE-treated glia cell does not differ from control (no drug treatment) astrocytes and this morphology is much different from the latrunculin A-treated cells reported in Fig. 1B. Micrographs were acquired with a confocal microscope; scale bar at 20 μm.

Continuous ethanol exposure (CEE) does not alter the viability of young or old days in vitro [DIV] cerebellar granule cells (CGCs). Phase-contrast photomicrographs of cultured young (5–12 DIV; top panels) and old (21–29 DIV; bottom panels) CGCs were obtained with a DXM1200F digital camera and NIS imaging software. Phase-contrast images of (A) control (12 DIV), (B) Day 5 (9 DIV) and (C) 12 DIV CGCs at the 72 + W time point indicate that all neurons are phase bright with extensive neuritic processes suggesting that CEE did not alter CGC viability. Results are similar for older DIV CGCs in that there are no apparent differences observed among (D) control (28 DIV), (E) Day 5 (25 DIV) or (F) 72 + W (28 DIV) CGC morphology and density; scale bar = 75 μm.

Continuous ethanol exposure (CEE) did not increase cell death of cerebellar granule cells (CGCs) expressed in young or old days in vitro [DIV] cultures. The percentage of dead cells as indicated by an uptake of the trypan-blue dye compared with nonblue cells was measured as presented in the Materials and Methods section. Paired t-test indicated no significant differences for young DIV CGCs at any time point post-CEE compared with culture-matched controls: t = 3.8, P = .7; t = 1.1, P = .3; t = 1.9, P = .1, and t = 1.6, P = .2 for Day 5 and for the 24 + W, 48 + W, and 72 + W time points, respectively. Similar results were observed for experiments conducted on old DIV CGCs: t = 0.6, P = .5 for cells at Day 5; t = 0.3, P = .7 at the 24 + W time point; t = 1.3, P = .3 at the 48 + W time point, and t = 1.0, P = .3 for the 72 + W time point. Summarized data for old DIV CGCs (A) and young DIV CGCs (B) are depicted as percent nonviable cells (cells that took up the dye and were therefore blue) compared with values from control cells.

Alterations in NMDA-induced currents (I_NMDA) of N-methyl-d-aspartate receptors (NMDARs) expressed in cerebellar granule cells (CGCs) depend on time post-continuous ethanol exposure (CEE). (A) Representative current traces acquired post-CEE from young days in vitro [DIV] CGCs are shown for Day 5, 24 + W, 48 + W, and the 72 + W time points along with current traces obtained from control cells. At Day 5, I_NMDA were smaller in receptors exposed to ethanol than I_NMDA acquired from receptors in CGCs never exposed to ethanol (control). Augmentation of receptor function due to ethanol withdrawal was shown by significant increases in I_NMDA compared with receptors in control CGCs for both the 24 + W (B) and 48 + W time points. By 4 days post-CEE or at the 72 + W time point, I_NMDA from NMDARs in ethanol-treated CGCs did not differ from currents acquired from receptors contained in control CGCs. Current traces were acquired from CGCs that were 6, 7, 8, and 9 DIV during the Day 5, 24 + W, 48 + W, and 72 + W time points, respectively. Control cells were from the same DIV and from the same culture batch. (B) Graph of summarized data depicting the percent change from control values; ***P ≤ .001.