Bicuculline induces epileptiform activity in slice cultures
In order to establish that neonatal mouse slice cultures treated with bicuculline develop spontaneous synchronized network activity we assessed the effect of acute bicuculline treatment on slice cultures grown in normal media. Slice cultures were made on P5 or 6 and following either 3 or 7 DIV, slice cultures were transferred to a recording chamber and perfused with normal ACSF followed by ACSF containing 50 μM bicuculline. shows representative extracellular field recordings. Under control conditions (normal ACSF) no spontaneous synchronized activity was observed on 3 DIV, but addition of bicuculline resulted in recurrent spontaneous epileptiform discharges in both area CA1 and CA3 in all slices examined (9 out of 9 slices). The mean burst duration and the inter-burst interval were nearly identical in the two areas (CA1:duration 0.72 ± 0.25 seconds; inter-burst interval 50.79 ± 50.66 seconds; CA3: duration 0.69 ± 0.16 seconds; inter-burst interval 49.72 ± 40.68 seconds). These results are in keeping with previous recordings from acute brain slices, where recurrent excitatory collaterals between CA3 pyramidal cells are thought to initiate discharging which then spreads to area CA1 through the Schaffer collateral pathway. At the 7 DIV time point, perfusion with bicuculline also resulted in robust recurrent spontaneous epileptiform discharges in both area CA1 and CA3 in each of 15 slices examined (data not shown). Again, mean burst duration and inter-burst interval did not differ significantly between CA1 (duration 1.20 ± 0.69 seconds; inter-burst interval 26.30 ± 14.82 seconds) and CA3 (duration 0.96 ± 0.25 seconds; inter-burst interval 20.35 ± 8.73 seconds). Discharges recorded at the 7 DIV timepoint were longer and more frequent than those recorded at the 3 DIV timepoint (ANOVA - duration: F(1,44) = 10.03, p ≤ 0.01, ANOVA - inter-burst interval: F(1,44) = 8.02, p ≤ 0.01). We have routinely recorded for over 1 hour after network discharging was initiated. Over this time, the characteristics of these events (frequency, duration and amplitude) remained quite stable.
Fig. 1 Representative traces of extracellularly recorded field activity in CA1 and CA3 from 3 DIV cultures with acute application of bicuculline. (A) Extracellular field recording from CA1 of a 3 DIV slice culture under control (CON) conditions in normal ACSF (more ...)
The length and branching complexity of dendritic arbors are reduced by chronic disinhibition
To determine if chronic disinhibition alters dendritic morphology, slices were treated from 3 DIV until 7 DIV with bicuculline. Individual CA1 pyramidal cells were then confocally imaged and basilar dendritic arbors reconstructed using Neurolucida. It was not possible to analyze apical dendrites since they crossed and overlapped with many other processes present in the apical dendritic layer. summarizes results from 3 separate experiments in which the dendrites from 18 control and 17 bicuculline-treated neurons were reconstructed. The bar graphs in show that following 4 days of chronic disinhibition a 40.6% decrease in dendritic length was observed (Control: 1736 ± 135.5 versus Bicuculline: 1031 ± 72.2 μm, p ≤ 0.001). In , the number of branch points in basilar dendritic arbors is shown to also be significantly decreased by 33.6% (Control: 17.4 ± 1.4 versus Bicuculline: 11.6 ± 1.6, p ≤ 0.01). This latter result led us to suspect that the reduction in dendritic length shown in may be solely the product of a decrease in the number of dendritic branches. However, this did not appear to be the case. In , the cumulative probability plot indicates that a higher percentage of dendritic segments are uniformly shorter after bicuculline treatment (p ≤ 0.01, Kolmogorov-Smirnov test)
. The bar graph in the inset shows that the average length of individual segments is significantly smaller (Control: 43.0 ± 1.7 versus Bicuculline: 36.7 ± 1.7μm, p
≤ 0.05). Terminal order segments of dendrites are thought to be sites where the majority of dendritic growth takes place (Rajan and Cline, 1998
). Based on this, we also examined alterations in the length of terminal order dendritic segments alone. Bicuculline treatment significantly decreased the length of terminal segments by 21% (Control: 52.2 ± 2.5 versus Bicuculline: 41.1 ± 2.4μm, p
≤ 0.001) - again suggesting that chronic disinhibition not only reduces the addition of new branches but also the growth of existing dendritic segments. shows maximum projection confocal images of representative neurons from the control () and bicuculline () treated groups. show Neurolucida reconstructions of the cell bodies and basilar dendrites of the cells in A and B and clearly illustrates that neurons treated with bicuculline have shorter dendritic arbors with fewer branches.
Fig. 3 Representative images of YFP positive CA1 pyramidal neurons and reconstruction of their dendrites from control and bicuculline treated slice cultures. Maximum projection confocal images of neurons from the control (A) and bicuculline (B) treated groups. (more ...)
At this point it seemed possible that the alterations in dendrite arbors induced by disinhibition might not result from network hyperexcitability but instead could be due to direct effects of GABAa receptor blockade. To address this possibility, we attempted to reproduce the results in by raising extracellular K+ to 12 mM to increase neuronal and network excitability. To establish that 12mM K+ was indeed able to induce synchronized network discharging in our slice cultures, extracellular field recordings were made while switching the chamber perfusate from ACSF containing 5.2mM K+ to 12 mM K+. In 5.2mM K+, synchronized discharging was not recorded. However, upon switching to 12mM K+ all slice cultures tested (DIV 3: 7 slices, DIV 7: 12 slices) displayed frequent (interevent intervals 0.32 to 2.5 sec) recurrent synchronized population discharges.
Based on these results, we examined the effects of a 4 day (DIV 3-7) high K+ treatment on dendrites. Results showed that as with bicuculline treatment the length of YFP-positive basilar dendrites was decreased (49%) by this alternative treatment (Control: 1188 ± 175.0 versus KCl: 606.7 ± 62.3 μm, p ≤ 0.05; n =8 and 7 respectively). In addition, the average length of individual segments was significantly smaller (Control: 43.3 ± 2.5 versus KCl: 27.8 ± 1.8μm, p ≤ 0.001), as was the length of terminal segments (Control: 57.6 ± 3.4 versus KCl: 33.3 ± 2.6μm, p ≤ 0.001). The number of dendritic branch points was also reduced 24.6% (Control: 11.8 ± 1.6 versus KCl: 8.9 ± 1.3μm). Since the effects of high K+ reproduced those of bicuculline, we concluded that the effects of bicuculline were mediated primarily by enhanced neuronal excitability.
Chronic disinhibition blocks the growth of dendrites
There are a number of ways in which chronic bicuculline treatment could reduce the length and branching complexity of dendritic arbors. One possibility is that network hyperexcitability damages the dendrites of developing pyramidal cells and reduces branching complexity by eliminating damaged segments or portions of segments. Typically, when dendrites are damaged by excitotoxic injury they display varicose swellings (Swann et al., 2000a). However, in the studies we have conducted thus far we have never observed such swellings in bicuculline-treated slice cultures. In keeping with these observations are other results from this in vitro
model in which neuronal cell death was not observed (Swann et al., 2007a
). Besides excitotoxic injury, another possible explanation for the observed stunted dendrites was that chronic disinhibition retarded the growth of dendrites. To address this possibility slice cultures were fixed at different times after initiation of bicuculline treatment. Control slices served to monitor the normal growth of dendrites in slice cultures. Results in show, that between 3 DIV and 7 DIV, the average length of the basilar dendrites doubled (1016.1 ± 80.1 versus 2128.7 ± 117.8 μm, p
≤ 0.001). However, at the same time basilar dendrites in bicuculline treated cultures failed to increase and actually decreased slightly in total length (1016.1 ± 80.1 versus 884.8 ± 109.1 μm, p
= 0.767). Indeed a two way ANOVA revealed that the effects of bicuculline treatment was significantly different from controls (F (1,76) = 67.7, p
≤ 0.001) as was the effects of time in culture (F (3,76) = 9.03, p
≤ 0.001). At 4, 5 and 7 DIV, the length of dendrites in bicuculline treated slices differed significantly from their control counterparts (p ≤ 0.05).
Fig. 4 Chronic disinhibition suppresses the growth of CA1 hippocampal pyramidal cell dendrites. (A-C) Between 3 and 7 days in vitro (DIV), the dendrites of CA1 pyramidal cells in control slices gradually increased in total length, number of branch points and (more ...)
shows concomitant alterations in basilar dendritic branch points. The number of branch points gradually increased in control slices between 3 and 7 DIV (3DIV: 12.1 ± 1.2 versus 7DIV: 19.2 ± 1.8, p ≤ 0.01). However, the number of branch points failed to increase and appeared to decrease during bicuculline treatment, but this seeming time-dependent reduction was not statistically significant (3DIV: 12.1 ± 1.2 versus 7DIV: 10.4 ± 1.6, p > 0.05, versus 4DIV: 9.2 ± 1.2, p > 0.05). However, at 4, 5 and 7 DIV, the number of dendritic branches in bicuculline treated slices differed from those of control slices (p ≤ 0.05). Again the effects of bicuculline treatment on the total length of dendrites could not be explained solely by the decrease in the number of dendritic branches since when the mean length of dendritic segments were compared in a difference in average segment length was observed at 7 DIV (7DIV Control: 48.9 ± 2.1 versus 7DIV Bicuculline: 36.7 ± 2.6 μm, p ≤ 0.001). However, unlike the effects on branch points this effect took several days to become apparent. The dendrite reconstructions shown in illustrate results summarized in . Between 3 and 7 DIV dendrites from pyramidal cells in control slices show a gradual but dramatic increase in overall length and branching complexity. These developmental changes are completely absent in bicuculline treated slice cultures.
A role for NMDA receptors in disinhibition-induced alteration in dendrites
Previous studies have implicated activation of NMDA receptors as an important contributor to activity-dependent dendritic growth (Rajan and Cline, 1998
;Wayman et al., 2006
). Thus we were motivated to determine if a persistent over-activation of NMDA receptors during network hyperexcitability plays a role in the cessation of growth observed in . The results of 3 such experiments are shown in . As before in these experiments, slice cultures were treated from day 3 in vitro
with bicuculline. A separate group was co-treated with bicuculline and 50μM d-APV. Controls consisted of untreated slices and a group treated with only APV. Slices were fixed on 7DIV. Results in show that as before bicuculline resulted in a decrease in the total length of basilar dendrites (Control: 1122.2 ± 112.3 versus Bicuculline: 656.3 ± 44.6 μm, p
≤ 0.001). But when slices were co-treated with bicuculline and APV the total length of the dendritic arbors was not statistically different from the control group (Control: 1122.2 ± 112.3 versus APV + Bicuculline: 1023.1 ± 75.1 μm, p
> 0.05) but was significantly larger than the bicuculline treated group (APV + Bicuculline: 1023.1 ± 75.1 versus Bicuculline: 656.3 ± 44.6 μm, p
≤ 0.0 01). This suggests that at least in terms of total dendritic length APV blocked the effects of bicuculline. A similar result was observed when dendritic branch points were examined (). Again, chronic bicuculline treatment resulted in a decrease in basilar dendritic branch points (Control: 13.6 ± 1.3 versus Bicuculline: 9.1 ± 0.7 μm, p
≤ 0.0 1). Dendritic branch number of pyramidal cells in slice cultures that were co-treated with bicuculline and APV were not statistically different from that of the control group (Control: 13.6 ± 1.3 versus APV + Bicuculline: 12.5 ± 1.2, p
> 0.05) but did differ significantly from cultures treated with bicuculline alone (p
≤ 0.01). This suggests that APV prevented the decrease in branch number that occurs during bicuculline treatment. APV alone appeared to increase the number of branch points but this also was not statistically significant (Control: 13.6 ± 1.3 versus APV: 17.4 ± 1.2 μm, p
> 0.05). Since APV rescued total dendritic length when applied with bicuculline, and had effects on branch number we next examined the effects of co-treatment on dendritic segment length. shows that APV abolished the effects of bicuculline on dendritic segment length. This suggests the suppression of dendrite segment growth that is observed in bicuculline treated slices (Control: 35.3 ± 1.4 versus Bicuculline: 29.6 ± 1.4 μm, p
≤ 0.0 1) is a NMDA receptor dependent process.
The dendritic reconstruction in supports the results shown in the bar graphs in . In these computerized tracings, bicuculline clearly diminishes the length and complexity of basilar dendrites and this effect appears to be abolished by APV. APV alone may increase dendritic arbor complexity in some cells and this could be reflected in the increased number of branch points shown in . However, as mentioned above, this effect was not statistically significant.
Signaling to CREB is altered
Calcium-dependent activation of the transcription factor CREB is thought to play a important role in activity-dependent growth of dendrites (Wong and Ghosh, 2002
). A number of signaling cascades including the CAM Kinase and MAP Kinase cascades appear to converge on CREB to mediate their effects (Redmond et al., 2002
;Vaillant et al., 2002
;Wayman et al., 2006
). Since our results suggest that dendritic growth is greatly diminshed by chronic disinhibition we next examined the effects of this treatment on activation of CREB by examining alterations in the phosphorylation at Ser-133. Immunohistochemistry was used to visualize pCREB in hippocampal slice cultures from YFP-H mice, since this allowed us to unequivocally examine pCREB expression in CA1 pyramidal cells. Results are shown in and were obtained on DIV 7, 4 days after initiating bicuculline treatment. In all slices that were treated with bicuculline a dramatic decrease in the expression of pCREB was observed (). Alterations in expression were not restricted to area CA1 since similar decreases in expression were observed in area CA3 and the dentate gyrus (data not shown). At higher magnification (), the nuclear localization of pCREB is evident in control slices - especially when merged with images of YFP. Most CA1 pyramidal cells do not express YFP in cultures from YFP-H mice. This contributes to the large number of pCREB and CREB positive cells that are YFP negative in these images (panels B, C, F and I).
Fig. 6 Chronic disinhibition reduces levels of phosphorylated CREB (pCREB) in CA1 hippocampal pyramidal cells. (A) Low magnification confocal image of a hippocampal slice culture showing typical pattern of immunohistochemical staining for pCREB. (B and C) Comparison (more ...)
Following chronic disinhibition, the level of pCREB expression is quite low (), compared to controls (). When the level of pCREB expression was quantified from images taken from 4 slices, we observed a 42% decrease in expression in area CA1 (Control: 89.0 ± 6.4 versus Bicuculline: 51.7 ± 2.6, F (1,6) = 29.4, p ≤ 0.01). Alterations in pCREB might be explained by a decrease in the expression of the CREB itself. However, this does not appear to be the case. When CREB expression was examined immunohistochemically no difference in expression were observed (). CREB was localized to nuclei but the level of expression was very similar in control and bicuculline treated slice cultures (). This was confirmed quantitatively since CREB expression showed no difference between the two condition (Control: 92.1 ± 6.7 versus Bicuculline: 87.9 ± 22.4, F(1,6) = 0.344, p = 0.58). These results suggest that activation of CREB by phosphorylation at Ser133 is reduced by chronic disinhibition.
Fig. 7 Expression of the transcription factor CREB is unaltered by chronic disinhibition. (A) Confocal image illustrating the typical pattern of CREB expression in a hippocampal slice culture. (B and C) Very similar CREB expression patterns are observed in hippocampal (more ...)