Article Figures & Data
Figures
- Figure 1.
Transfection with rAAV8/hSyn-mCherry-Cre in an fCaNB1 mouse slice cultures results in a reduction in CaNB expression. A, B, Low-magnification confocal images of area CA1 showing the transfection of a large number of neurons. mCherry (shown here as green) which is fused to Cre serves as a reporter of viral transfection. C, Image illustrates the typical widespread distribution of CaNB in area CA1 from wild-type mice. The protein is highly expressed in basal (stratum oriens, SO) and apical (stratum radiatum, SR) dendritic layers. E, Area in C outlined by box and shown at higher magnification. Notable is the lack of immunoreactivity in cell nuclei, particularly in the cell bodies of stratum pyramidale (SP). D, AAV-Cre transfection of fCaNB1 mouse cultures resulted in a marked reduction in CaNB1 immunoreactivity. F, Area in D outlined by box and at higher magnification. Intermixed with transfected cells are untransfected neurons (see arrowheads) in which the cytoplasm and somas and apical dendrites stain intensely for CaNB1. The staining of these untransfected neurons stand out against a background of immunonegative transfected neurons. Tissue was processed on DIV8. Scale bars, 100 µm (A–D) and 300 µm (E, F).
- Figure 2.
AAV/hSyn-mCherry-Cre transfections result in a marked reduction in CaNB1 expression in transfected neurons from fCaNB1 mice but had no effect of CaNB1 expression in neurons from wild-type (WT) mice. NeuN immunoreactivity was used to stain neuronal cytoplasm and nuclei. Nuclear mCherry reports viral transfection and CaNB1 immunoreactivity is shown in red. A, Transfected neurons in slices from wild-type mice showed no discernable effect on CaNB1 expression. Arrows point out two untranfected cells. B, In contrast, viral transfections in slices from fCaNB1 mice resulted in a complete loss of CaNB1 immunoreactivity which is markedly different from the immunoreactivity of untransfected cells (arrows). C, Quantification of results from 10 wild-type and 8 fCaNB1 mouse slices showed that over 90% of transfected cells from fCaNB1 mice had no detectable CaNB1 in their cytoplasm but virtually every transfected neuron from wild-type cells had high levels of CaNB1. Scale bar, 50 µm; ***α = 0.001.
- Figure 3.
Acute treatment with the bicuculline results in a dramatic nuclear translocation of the CREB transcriptional coactivator CRTC1. A, Under baseline conditions, CRTC1 is cytoplasmic and largely excluded from neuronal nuclei which are immunostained for NeuN. However, 3 h after initiating treatment with bicuculline (BIC 100 µM), CRTC1 has shuttled to the nuclei of virtually every neuron in this field of view. B, The rapid time course of CRTC1 nuclear translocation. Here, the ratio of the intensity of nuclear to cytoplasmic CRTC1 immunostaining is computed at 0 min, 5 min, 30 min, 1 h, 3 h, and 8 h of bicuculline treatment. The graph summarizes results from three separate experiments, three slices per experiment, thus, n = 9 for each of the six time points. Scale bar, 100 µm; ***α = 0.001.
- Figure 4.
mCherry-Cre viral transfections of neurons in fCaNB1 mouse slices prevents the nuclear translocation of CRTC1. DAPI is used to stain nuclei while mCherry (shown here in green) reports neuronal transfections. A1–A8, Under control (CON) conditions in both wild-type (WT) and fCaNB1 slices, CRTC1 is cytoplasmic and excluded from nuclei. A9–A12, In slices from wild-type mice, bicuculline (BIC) treatment (100 µM for 4 h) resulted in CRTC1 nuclear translocation whether the neuron was transfected or not. To illustrate, five cells are circled in A9. Two of which were transfected (A10) and CRTC1 was translocated to the nuclei of all five cells (A11, A12). A13–A16, In contrast are results from fCaNB1 mice, where bicuculline treatment did not induce a CRTC1 nuclear translocation in transfected cells but did so in untransfected cells. Five representative cells are circled in A13. Three of these were transfected (A14). Bicuculline treatment resulted in the nuclear translocation of CRTC1 in the two untransfected cells but not in the three transfected neurons (A15 and A16). B, The computed ratio of the intensity of nuclear to cytoplasmic CRTC1 immunostaining was significantly increased in transfected neurons from wild-type mice but was not altered in transfected cells from fCaNB1 mice. The bar graphs summarize results from three separate experiments, nine slices total for each of the four conditions. Scale bar, 50 µm; *α = 0.05; ***α = 0.001.
- Figure 5.
Elimination of CaNB from hippocampal pyramidal cells does not impair their participation in seizure activity. A, Multiphoton image obtained during a physiology recording session. B, Simultaneous local field potential (LFP) recordings and cell attached patch recordings (Cell) of seizure-like activity induced by bicuculline (50 µM). The cellular recordings were obtained from the centermost GFP-positive pyramidal cell in A. This cell lacks CaNB, since it has been virally transduced and expresses mCherry in its nucleus (seen here as yellow). Recordings suggest the spiking in the cell occurs nearly simultaneous with the synchronous discharging of the population. C, This correlation is verified by a cross-correlation analysis of population spikes with spikes in individual pyramidal cells in four separate experiments. Yellow dashed line indicates when population spikes occurred. The large peak centered on 0 ms shows that the pyramidal cells usually fired within a few milliseconds before or after the discharge of the population. When the time stamp for every cell action potential was randomly assigned a time during the field potential recordings no correlation was observed (red overlay), and this differed significantly from the original data. Stars indicate significant (p ≤ 0.05) differences between original and randomized datasets at the indicated time points. Scale bar, 25 µm.
- Figure 6.
Elimination of CaNB1 from CA1 hippocampal pyramidal cells prevents electrographic seizure-induced reductions of basilar dendrite length and branch number. A, B, Treatment of slice cultures from wild-type (WT) mice for 2 d with bicuculline (BIC 100 µM) results in a decrease in both dendrite length and branch number in mCherry-Cre-transfected neurons. However, an identical treatment of slice cultures from fCaNB1 mice failed to reduce dendrite length or branch number. C, Results of Sholl analyses are consistent with the results in A, B, i.e., dendritic arbors were reduced in length and branching complexity following treatment of transfected neurons from wild-type mice with bicuculline but this effect was greatly attenuated in transfected pyramidal cells from fCaNB1 mice. D, Representative dendrite reconstructions from the four experimental groups. Nine pyramidal cells were reconstructed for each condition. Scale bar, 100 µm; *α = 0.05.
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