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Rapid and reversible changes in dendrite morphology and synaptic efficacy following NMDA receptor activation: implication for a cellular defense against excitotoxicity

Yuji Ikegaya^1,*,, Jeong-Ah Kim^1,*, Minami Baba², Takeshi Iwatsubo², Nobuyoshi Nishiyama¹ and Norio Matsuki¹

¹ Laboratory of Chemical Pharmacology and
² Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
^* These authors contributed equally to this work

View larger version (40K): [in a new window]   Fig. 1. NMDA induced neuronal cell death in a concentration- and time-dependent manner. (A) Confocal fluorescence micrographs show hippocampal CA1 pyramidal cells double-labeled with DiO (green) and PI (red) 24 hours after 10 minutes exposure to vehicle (a) or 30 µM NMDA (b). The plasma membrane was severely damaged in a PI-positive neuron and thereby the intracellular membrane compartments were also stained with DiO, whereas only the plasma membrane was labeled with DiO in a PI-negative neuron. The faint red signal in the background is derived from an autofluorescence of the cytoplasm of pyramidal neurons. (B) PI fluorescence was imaged in the hippocampal slices 24 hours after 10 minutes exposure to vehicle (a), 30 µM NMDA (b), or a combination of 30 µM NMDA and 10 µM MK-801 (c). (C) Twenty four hours after treatment with NMDA at concentrations in the range of 1-100 µM for 10 minutes, the fluorescence intensity of PI was quantified in each hippocampal subregion, i.e. the CA1 region (circle), the CA3 region (triangle) and the DG (square). Massive neuronal death was observed mainly in the CA1 region at 30 µM NMDA but not evident in any subregions at concentrations of less than 10 µM. (D) NMDA (3 or 30 µM) was applied for 10 minutes in the absence or presence of 10 µM MK-801. MK-801 was added to culture medium 30 minutes before NMDA exposure. Twenty four hours after NMDA exposure, PI fluorescence was measured in the CA1 region. NMDA-induced neuronal death was prevented by MK-801. (E) PI fluorescence intensities in the CA1 region (circle), the CA3 region (triangle) and the DG (square) were measured 0, 24, 48, 72, 120 or 168 hours after 10 minutes exposure to 30 µM NMDA. Cell death was detected only at 24 and 48 hours. **P<0.01 versus Control, ##P<0.01 versus 30 µM NMDA. Data are the means±s.e.m. of eight to ten slices.

View larger version (113K): [in a new window]   Fig. 2. Rapid formation of dendritic focal swelling in response to NMDA exposure. (A) Time-lapse confocal images of an apical dendrite of DiI-labeled CA1 pyramidal neuron following 10 minutes exposure to 30 µM NMDA. Numbers above the images indicate time (minutes) after NMDA treatment. NMDA induced a decrease in the number of spines and subsequently produced focal swelling along the dendrites. (B) Electron micrograph of varicosities in apical dendrites of DiI-labeled CA1 pyramidal neurons in untreated (a) and NMDA-treated (30 µM, 10 minutes) (b) slices. The varicosities, indicated by arrowheads, contained clear vacuoles (V) and many short fragments of degenerated microtubules.

View larger version (29K): [in a new window]   Fig. 3. Characterization of NMDA-induced morphological changes in dendrites. (A) Immediately after 10 minutes exposure to NMDA at concentrations in the range of 1-100 µM, slices were fixed with 4% paraformaldehyde and the dendrites were labeled with DiI. The density of spines along the length of the dendrites (a), the percentage of dendrites bearing varicosities (b), the density of varicosities along the dendrites (c) and the average size of varicosities (d) were measured. NMDA treatment resulted in a reduction in the spine density and simultaneously caused an increase in the number and size of varicosities in a concentration-dependent manner. No regional difference in these morphological changes was detected among the CA1 region (circle), the CA3 region (triangle) and the DG (square). (B) Slice cultures were fixed after 4, 6 and 10 minutes of NMDA exposure, or at 6, 12, 24, 48, 96 and 144 hours after 10 minute exposure to 3 µM NMDA and the ratio of varicosity-bearing dendrites was measured. The dendritic varicosities appeared within 4 minutes of exposure and were observed in about 80% of dendrites by 6 minutes of exposure, but gradually decreased until 144 hours after removal of NMDA. (C) NMDA (3 or 30 µM) was applied for 10 minutes in the absence or presence of 10 µM MK-801. Immediately, the cultures were fixed and the ratio of dendrites with varicosities was measured in the CA1 region. MK-801 was added to culture medium 30 minutes before NMDA exposure. NMDA-induced varicosity formation was almost completely prevented by MK-801. **P<0.01 versus Control, ##P<0.01 versus slices treated with corresponding doses of NMDA. Data are the means±s.e.m. of 9-12 slices.

View larger version (25K): [in a new window]   Fig. 4. Discrepancies between neuronal death and dendritic varicosity formation. Slices were treated with kainate (A), AMPA (B) or other drugs (C). Kainate, AMPA and veratridine (10 µM) were applied for 10 minutes. Ca2+-free buffer was applied for 30 minutes. Colchicine (100 µM), cytochalasin D (1 µM) and latrunculin A (1 µM) were applied for 12 hours. The cultures were divided into two groups: one group was cultivated at 37°C for 24 hours and PI uptake was measured (a), and the other group was immediately fixed with 4% paraformaldehyde and the ratio of dendrites bearing varicosities was measured (b). Panel C shows values in the CA1 region. Varicosity formation was induced by various stimulants, even at low concentrations that did not give rise to cell death. **P<0.01 versus Control. Data represent the means±s.e.m. of 9-13 slices.

View larger version (48K): [in a new window]   Fig. 5. NMDA-induced depletion of cell-surface AMPA receptors. Slices were treated with 3 µM NMDA for 10 minutes and immediately fixed with 4% paraformaldehyde and then the immunohistochemical localization of the GluR1 subunit was analyzed with a confocal microscope. (A) Representative confocal sections through the CA1 stratum pyramidale (SP) and the stratum radiatum (SR) show the cellular distribution of GluR1. AMPA receptors containing GluR1 on the cell surface were labeled with antibody under nonpermeant conditions (Surface GluR1) and the total GluR1 population was probed under permeant conditions (Total GluR1). (B) NMDA-induced reduction of cell-surface GluR1 was assessed in the CA1 stratum pyramidale (a) and the CA1 stratum radiatum (b) by using quantitative colorimetric assay. NMDA induced a decrease in cell-surface GluR1 subunits (Surface) without affecting the total number of GluR1 (Total), resulting in a lower proportion of surface AMPA receptors. The GluR1 internalization was predominantly observed in the dendrite-rich area (SR), as compared with the soma-rich zone (SP). *P<0.05 versus Control. Data represent the means±s.e.m. of eight slices.

View larger version (21K): [in a new window]   Fig. 6. Rapid, long-lasting depression of synaptic transmission following NMDA exposure. (A) Time course of changes in fEPSP amplitude following a 10 minute bath application of 3 µM NMDA. fEPSP evoked by stimulation of the Schaffer collaterals was recorded extracellularly from the CA1 stratum pyramidale of cultured hippocampal slices. Representative fEPSP recordings at times -10 (1) and 58 (2) are shown in the inset. Arrows indicate stimulation-elicited artifacts. Data are expressed as the percentage of pretreatment fEPSP amplitude; means±s.e.m. of four slices. (B) Reversible NMDA-induced suppression of synaptic transmission. fEPSP was recorded immediately before (pre) or at 0, 1, 24 and 48 hours after 10 minutes exposure to 3 µM NMDA. Data are the means±s.e.m. of seven to eight different slices. *P<0.05, **P<0.01 versus Pre. NMDA induced a rapid, long-lasting decrease in synaptic response, which almost completely recovered after NMDA washout. Recovery time was relatively slow, requiring 48 hours.

View larger version (48K): [in a new window]   Fig. 7. Low Na+ buffer, ethacrynic acid and protease inhibitors prevent NMDA-induced dendritic varicosity formation. (A) Representative confocal images of dendrites of CA1 pyramidal cells in untreated slices (a) or 30 µM NMDA-treated slices in normal (b) or low Na+ solution (c). Culture medium was changed to low Na+ buffer 30 minutes before 10 minutes exposure to NMDA. No varicosity was generated under low Na+ conditions. (B) Effects of various manipulations and drugs on NMDA-induced varicosity formation. The culture medium was changed to lowered Na+ solution or 200 mM sucrose-containing (hyperosmotic) medium 30 minutes before NMDA exposure. Tetrodotoxin (1 µM), HgCl2 (100 µM), amiloride (100 µM), furosemide (100 µM) and ethacrynic acid (400 µM) were added 30 minutes before NMDA exposure. A mixture of protease inhibitors (1:1000) was applied 12 hours prior to NMDA exposure. Slices received 10 minutes treatment with 0 µM (open columns) or 30 µM NMDA (filled columns), immediately followed by fixation with 4% paraformaldehyde. Low Na+ buffer, ethacrynic acid and protease inhibitors significantly blocked the varicosity formation, whereas tetrodotoxin, amiloride and furosemide induced varicosity formation. Data are expressed as the means±s.e.m. of 9-12 slices. **P<0.01 versus intact slices (None and Control), ##P<0.01 versus NMDA alone (None and NMDA).

View larger version (38K): [in a new window]   Fig. 8. Inhibition of varicosity formation aggravates NMDA-induced neuronal death. (A) Representative confocal images of PI fluorescence in hippocampal slices 24 hours after 10 minutes exposure to 3 µM (a,b) or 30 µM (c,d) NMDA in normal (a,c) or low Na+ conditions (b,d). Culture medium was changed to normal or low Na+ buffer 30 minutes prior to NMDA treatment. (e) PI fluorescence intensity was quantified in the CA1 region (solid columns), the CA3 region (hatched columns) and the DG (open columns). Low Na+ conditions exacerbated NMDA excitotoxicity; NMDA in low Na+ solution evoked neuronal death all over the hippocampus. Note that treatment with NMDA even at a concentration of 3 µM showed apparent neurotoxicity. (B) Slices were treated with a mixture of protease inhibitors (1:1000) for 12 hours and exposed to 3 or 30 µM NMDA. They were subsequently incubated in normal medium at 37°C for 24 hours and PI uptake was measured. Treatment with protease inhibitors exacerbated NMDA-induced neuronal death. **P<0.01 versus Control, #P< 0.05, ##P<0.01 versus corresponding concentrations of NMDA. Data are the means±s.e.m. of 9-12 slices.

View larger version (19K): [in a new window]   Fig. 9. Lack of effect of NMDA on synaptic transmission in low Na+ solution. When Na+ concentration of the bath solution was reduced from 150 mM to 30 mM, fEPSP amplitudes dropped markedly within 10 minutes, but completely recovered within 30 minutes after application of basal Na+ concentration (open circles). Bath application of 3 µM NMDA for 10 minutes did not cause depression of fEPSP amplitudes in low Na+ solution (closed circles). Data are expressed as a percentage of baseline fEPSP amplitude (at time -40); means±s.e.m. of four slices.