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Supplement to: Glutamate receptors regulate actin-based plasticity in dendritic spines by M Fischer, S Kaech, U Wagner, H Brinkhaus and A Matus Nature Neuroscience, September 2000, Volume 3 Number 9 pp 887 - 894
Glutamate blocks actin dynamics in dendritic spines. This Time Movie-lapse sequence shows GFP-actin dynamics in part of the dendrite shown in Fig. 1. The first section was recorded under control conditions followed by a sequence recorded after 100 µM glutamate had been added to the medium together with 100 µM APV. The high level of actin dynamics seen in these spines under control conditions is typical of all cells maintained in low density culture.
AMPA blocks spine motility in the presence of NMDA receptor antagonists. Effect of AMPA in the presence of MK801. There are two versions of this sequence, Fig2a_1.mov showing the original low magnification recording of a long stretch of dendrite and Fig2a_2.mov showing a small extract of the same sequence at higher magnification. The effects of AMPA both alone and in the presence of the NMDA receptor channel blocker MK801 (10 µM) are documented in this single recording. The first half of this sequence, showing the reversible effect of AMPA on a long stretch of dendrite, was used to prepare the difference images shown in Fig. 5. The sequence shows: 1. (no label) the control condition, when spines are motile; 2. (labeled "+AMPA") a brief period in which the medium was switched to one containing 2 µM AMPA, when spine motility was blocked. Note the rounding up of the spine heads; 3. (no label) an intermediate period when the AMPA was washed out to demonstrate that spine motility recovers; 4. (labeled "+MK801") after the AMPA receptor antagonist MK801 was added to the medium, when spine motility continued unaffected; 5. (labeled +AMPA + MK801) after AMPA was added to the medium in addition to the MK801. During this long final period spine motility was inhibited, showing that it is produced by AMPA receptor stimulation independently of the blocked NMDA receptors.
Figure 2b: Effect of AMPA in the presence of APV. This sequence focuses on part of a dendrite bearing spines with a variety of characteristic morphologies including long necked, mushroom shaped and stubby. The two extracts are from a single long experiment, the first recorded under control conditions, the second in the presence of 1 µM AMPA. 100 µM APV was present throughout the recording to block NMDA receptors. Spines of all categories show actin-based motility under control conditions which is blocked by AMPA. Note that, as in the example shown in Fig2a_2.mov, the effect of AMPA receptor activation is to inhibit the formation of actin-rich protrusions from the spine head.
AMPA blocks spine actin dynamics in organotypic slice cultures. Three video sequences, taken from a time-lapse recording of the 4 week-old slice culture shown in the still frames in the top row of Fig. 3. The sequences are labeled as follows: Fig3_1.mov, spine dynamics in the control condition; Fig3_2.mov recorded in the presence of 1µM AMPA; Fig3_3.mov after washout of the AMPA. Each sequence represents 15 min of recording comprising 60 frames taken at 15 sec intervals. Note the lack of spine dynamics during the sequence shown in Fig3_2, indicating that spine movement seen under control conditions is due to actin dynamics and not focus shift or other artifacts of the recording technique.
Blockade of spine motility by NMDA requires lowering Mg2+ in the medium. This recording begins with a period of control recording followed by a sequence in which 1 µM NMDA was added in regular medium containing 0.5 mM Mg2+ (labeled "+NMDA") which did not inhibit spine motility. The medium was then switched to one containing NMDA and 0.1 mM Mg2+ (labeled +NMDA + low Mg2+) when spine motility was inhibited.
Figure 6. AMPA receptor blockade of spine motility is Na+ dependent. A single time-lapse recording during which the following conditions were applied: 1. (labeled "Control") in control medium the spines on this segment of dendrite were motile; 2. (labeled "-Na") when NaCl was replaced by choline chloride spine motility continued undiminished; 3. (labeled "-Na, +AMPA") subsequently 200nM AMPA was added to the medium but still in the absence of sodium. Despite the presence of AMPA, motility was not noticeably inhibited when Na+ was absent; 4. (labeled "+Na+AMPA") When NaCl was added back to the medium AMPA now blocked spine motility.
Additional data. Blockade of voltage dependent Na+ channels does not affect AMPA-induced inhibition of spine motility. The first part of this recording shows that spine motility remains intact in a cell that had been exposed to 1µM TTX for 2 hours. The second part of the recording shows that adding AMPA (2 µM) still inhibited spine motility even though TTX was present. This particular cell had a well-developed set of recurrent axons which are visible among its dendrites as thinner processes containing faint patches of actin. These are seen to travel along the axons during the recording and this transport is not blocked by AMPA treatment.
Figure 7. Raising [K+]o induces transitory blockade of spine motility. At the time indicated by the appearance of the label "KCl" the external K+ concentration was raised to 8 mM. This produced a transitory inhibition of spine motility and rounding up of spine heads, consistent with the spontaneous recovery of the membrane potential from the temporary depolarization induced by raising the external K+ concentration.
Supplementary data_cd. Voltage-activated calcium channel (VAC) antagonists and AMPA-induced inhibition of spine motility. Fig7_Ni: These 4 excerpts are taken from a single continuous time-lapse recording under the following conditions: 1) a period of control recording in regular Tyrode's solution to establish the base level of motility. 2) After the addition of 100 µM Ni2+, which does not influence spine motility. 3) With the addition of 2 µM AMPA in the continued presence of 10 µM Ni2+. The usual inhibition of motility by AMPA is suppressed by the Ni2+. 4) After changing the medium to one still containing AMPA but now lacking Ni2+. Spine motility is now shows AMPA inhibition. APV (100 µM) to block NMDA receptors was present in all solutions.
Supplementary data_ni. The equivalent experiment to the one described above showing that AMPA can inhibit spine motility in the presence of 500 µM Cd2+. Supplementary data_nif. Showing that the HVAC antagonist nifedipine (20 µM) does not suppress AMPA inhibition of spine motility.
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