(A and B) Normalized average FRAP signal over time in untreated neurons (blue) and the same neurons after (A) 100 µM glutamate, or (B) 100 µM NMDA (orange). Photobleaching was performed between the arrows. Time = 0 was set to when photobleaching ends and fluorescence starts to recover. Error bars are standard error of the mean (SEM). n = 19 for glutamate; n = 17 for NMDA. (C) The ER within the ROI (dashed line) was modeled as consisting of two volumes: V₁ and V₂. The RedER molecules move within these volumes with rate constants k₁ and k₂ respectively. (D and E) Box plot of τ_eff values of the same neurons shown in A and B. Untreated neurons are blue and the same neurons after (D) 100 µM glutamate or (E) NMDA are orange. Note the difference in scale between D and E. The line in the box is the median and the box represents the 25–75 percentiles. Whiskers extend to the extreme values as long as they are within a range of 1.5× box length. Values outside this range are plotted as outliers. avg.: average.

(A) Hippocampal expression of RedER in Thy1-RedER transgenic mouse line 18. (B) RedER expression in line 27. The expression pattern of the transgene differs slightly in that line 18 has no expression in pyramidal cells of CA3 but does express at high level in the hilus. (C) Representative images of dendritic ER structure in a CA3 pyramidal cell from line 27 with continuous ER prior to any treatment. (D) 100 µM NMDA triggered rapid ER fission. (E) 25 µM MK-801 led to fusion within 25 min. (F) Normalized average FRAP signal over time in untreated neurons (blue), the same neurons after NMDA for 10 min (orange) (note that MK-801 was added after 5 min) and after MK-801 (purple). Photobleaching was performed between the arrows. Time = 0 was set to when photobleaching ends and fluorescence starts to recover. Error bars are SEM. n = 4 neurons in 4 slices. Scale bars: 500 µm in A and B, 10 µm in C-E. avg.: average.

(A) Three representative images of CA1 and CA3 somata with normal ultrastructure. RER membranes studded with ribosomes are indicated with arrows. (B) The ultrastructure after 100 µM glutamate for 5 min. No normal RER profiles can be seen; instead the cytosol contains numerous dilated vesicles or tubules/cisterns that appear to have fewer ribosomes attached. Scale bar in all panels 500 nm.

(A) Representative image of a dendrite from a neuron with normal dendritic and ER structure. (B) Treatment with 5 µM ionomycin for 10 min caused ER fission along with a pronounced effect on gross dendritic structure (dendritic blebbing). (C) Normalized average FRAP signal over time in untreated neurons (blue) and the same neurons after ionomycin for an average of 35 min (orange). Photobleaching was performed between the arrows. Time = 0 was set to when photobleaching ends and fluorescence starts to recover. Error bars are SEM. n = 18. (D) Box plot of τ_eff values in untreated neurons (blue) and the same neurons after ionomycin (orange). The line in the box is the median and the box represents the 25–75 percentiles. Whiskers extend to the extreme values as long as they are within a range of 1.5× box length. (E) Representative image of a dendrite from a neuron with normal dendritic and ER structure. (F) Treatment with 25 µM MK-801 for 20 min prior to 5 µM ionomycin prevented ER fission and only caused minor effects on gross dendritic structure. (G) Normalized average FRAP signal over time in untreated neurons (blue) and the same neurons after MK-801 and ionomycin for an average of 50 min (orange). Photobleaching was performed between the arrows. Time = 0 was set to when photobleaching ends and fluorescence starts to recover. Error bars are SEM. n = 18. (H) Box plot of τ_eff values in untreated neurons (blue) and the same neurons after ionomycin (orange). The line in the box is the median and the box represents the 25–75 percentiles. Whiskers extend to the extreme values as long as they are within a range of 1.5× box length. One neuron was outside this range and plotted as an outlier. Scale bar in all panels: 10 µm. avg.: average.

(A) Representative image of a dendrite of a living hippocampal neuron transfected to express cytosolic EGFP and RedER showing normal ER morphology. (B) When exposed to 100 µM glutamate, rapid fission of the ER occurred after 1 min. Note the lack of change in dendritic and dendritic spine morphology in the green channel. (C) Image of proximal dendrite exposed to 100 µM glutamate for 20 min clearly showing the fragmented appearance of the ER in the xy (upper panel) and xz (lower panel) dimensions. (D) Representative image of a dendrite with normal morphology. (E) When exposed to 100 µM NMDA, rapid fission of the ER occurred within 3 min. Note the lack of change in dendritic and dendritic spine morphology in the green channel. Scale bar in all panels: 10 µm.

(A) Representative image of a dendrite from a neuron with normal dendritic and ER structure. (B) 100 µM of NMDA caused rapid ER fission without any effect on gross dendritic structure. (C) Antagonizing NMDA receptor activation by 25 µM MK-801 allowed for ER fusion and recovery of ER structure by 24 h. (D) Normalized average FRAP signal over time in untreated neurons (blue), the same neurons after NMDA for an average of 20 min (orange) (note that MK-801 was added after 2–5 min) and after MK-801 for 24 h (purple). Photobleaching was performed between the arrows. Time = 0 was set to when photobleaching ends and fluorescence starts to recover. Error bars are SEM. n = 20. (E) Box plot of τ_eff values in untreated neurons (blue) and the same neurons after NMDA (orange) and MK-801 (purple). The line in the box is the median and the box represents the 25–75 percentiles. Whiskers extend to the extreme values as long as they are within a range of 1.5× box length. Scale bar in all panels: 10 µm. avg.: average.

(A) Three representative images of dendrites from CA1 and CA3 in organotypic slices. ER profiles are indicated by arrows. (B) The ultrastructure after 100 µM glutamate for 5 min before fixing. No normal SER profiles were seen; instead the dendritic cytosol contains numerous dilated vesicles indicated by arrows. Boxed areas in the low magnification panel are enlarged below. Scale bar in all panels 500 nm.

(A) Representative image of a dendrite from a neuron with normal dendritic and ER structure. (B) Treatment with 25 µM MK-801 for 10 min prior to 100 µM glutamate prevented ER fission although an effect on gross dendritic structure was seen (reduction in spine length). (C) After 24 h, 19 out of 20 cells had survived and resumed normal morphology. (D) Normalized average FRAP signal over time in untreated neurons (blue), the same neurons after MK-801 and glutamate for an average of 35 min (orange) and after 24 h (purple). Photobleaching was performed between the arrows. Time = 0 was set to when photobleaching ends and fluorescence starts to recover. Error bars are SEM. (E) Box plot of τ_eff values in untreated neurons (blue) and the same neurons after glutamate (orange). The line in the box is the median and the box represents the 25–75 percentiles. Whiskers extend to the extreme values as long as they are within a range of 1.5× box length. One neuron was outside this range and plotted as an outlier. Scale bars in all panels: 10 µm. avg.: average.

Rather than viewing the ER as having two static structural states (continuous and fragmented) the ER membranes may be undergoing constant fission and fusion events. In the resting state fusion events balance fission events. After NMDA receptor stimulation this balance may be shifted so that fusion events are more rare than fission events.