Long-Term Potentiation in Isolated Dendritic Spines

BACKGROUND:In brain, N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation can induce long-lasting changes in synaptic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor (AMPAR) levels. These changes are believed to underlie the expression of several forms of synaptic plasticity, including long-term potentiation (LTP). Such plasticity is generally believed to reflect the regulated trafficking of AMPARs within dendritic spines. However, recent work suggests that the movement of molecules and organelles between the spine and the adjacent dendritic shaft can critically influence synaptic plasticity. To determine whether such movement is strictly required for plasticity, we have developed a novel system to examine AMPAR trafficking in brain synaptosomes, consisting of isolated and apposed pre- and postsynaptic elements. METHODOLOGY/PRINCIPAL FINDINGS:We report here that synaptosomes can undergo LTP-like plasticity in response to stimuli that mimic synaptic NMDAR activation. Indeed, KCl-evoked release of endogenous glutamate from presynaptic terminals, in the presence of the NMDAR co-agonist glycine, leads to a long-lasting increase in surface AMPAR levels, as measured by [(3)H]-AMPA binding; the increase is prevented by an NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5). Importantly, we observe an increase in the levels of GluR1 and GluR2 AMPAR subunits in the postsynaptic density (PSD) fraction, without changes in total AMPAR levels, consistent with the trafficking of AMPARs from internal synaptosomal compartments into synaptic sites. This plasticity is reversible, as the application of AMPA after LTP depotentiates synaptosomes. Moreover, depotentiation requires proteasome-dependent protein degradation. CONCLUSIONS/SIGNIFICANCE:Together, the results indicate that the minimal machinery required for LTP is present and functions locally within isolated dendritic spines

Ultrastructural and biochemical characterization of synaptosomes.

<p>A, Low power magnification of crude synaptosomes prepared from mouse brain showing pre-synaptic elements (pre) containing synaptic vesicles (SV) as well as post-synaptic elements (post), free mitochondria (M) and other unidentified structures. Scale bar corresponds to 0.5 µm. B, High power views of intact, sealed and tightly apposed pre- and post-synaptic elements, characteristic of asymmetric glutamatergic synapses. Clearly identifiable electron dense PSDs can be observed beneath the post-synaptic plasma membrane. The post-synaptic element also contains tubular and vesicular structures (TV) within the cytosolic compartment. Mitochondria could also be observed in the pre-synaptic terminal (pre+M). C, Fractionation of synaptosomes (P2) into synaptic plasma membrane- (LP1), synaptic vesicle- (LP2) and PSD-enriched fractions. Subcellular fractions were immunoblotted with antibodies against NMDA and AMPA receptor subunits (NR1 and GluR2, respectively) as well as markers of postsynaptic density (PSD-95), endosomal (EEA1, Rab11), lysosomal (LAMP2) and synaptic vesicle (Synaptophysin) compartments.</p

Model of LTP and AMPA-mediated depotentiation in synaptosomes.

<p><i>Synaptic NMDAR stimulation</i>, High KCl concentration depolarizes synaptosomes releasing endogenous glutamate, which activates synaptic NMDARs in conjunction with the NMDAR co-agonist glycine. Both glutamate and glycine cooperate to open receptor channels, facilitating calcium influx into synaptosomes, which, in turn, initiates a cascade of events resulting in the translocation of AMPARs from internal pools into synaptic sites. <i>AMPAR stimulation</i>, After LTP, AMPA depotentiates synaptosomes by translocating AMPARs from synapses into internal pools.</p

Proteasome function is required for AMPA-induced depotentiation of synaptosomes.

<p>A, Proteasome inhibitors block AMPA-induced AMPAR internalization after LTP in synaptosomes. LTP was induced in synaptosomes and AP5 (100 µM) was added as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006021#pone-0006021-g002" target="_blank">figure 2F</a>. After 10 min., samples were treated with either the proteasome inhibitor MG-132 (50 µM), lactacystin (10 µM) or control buffer, followed by AMPA (100 µM) for 30 min. to depotentiate synaptosomes as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006021#pone-0006021-g004" target="_blank">figure 4B</a>. Both proteasome inhibitors completely blocked the AMPA-induced reduction surface AMPAR levels, as determined by specific binding of [³H]-AMPA to non-permeabilized synaptosomes. Values represent means±SEM of 3 independent experiments. Dunnett's test was used to compare proteasome inhibitor vs. control synaptosomes. *, p<0.05. B and C, AMPA-induced depotentiation of synaptosomes leads to the proteasome-dependent degradation of AMPAR scaffolding protein (C) but not AMPAR subunits (B). Synaptosomes were depotentiated after LTP by incubating with AMPA (100 µM) for the indicated times as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006021#pone-0006021-g004" target="_blank">figure 4B</a>. The samples were then immunoblotted with indicated antibodies against the GluR AMPAR subunits (B) or against the AMPAR adaptor proteins GRIP1, GRIP2 and PICK1 (C). Anti-Erk was used as a loading control. Only GRIP1 and GRIP2 levels decreased in response to AMPA-induced depotentiation. The decrease was blocked by MG-132.</p

AMPA depotentiates synaptosomes.

<p>A, Synaptosomes were either left untreated (0 and 60 min. time points) or treated with AMPA (100 µM) for the indicated times. B, LTP was induced in synaptosomes and AP5 (100 µM) was added as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006021#pone-0006021-g002" target="_blank">figure 2F</a>. After 10 min., samples were either left untreated (0 time point) or treated with AMPA (100 µM) for the indicated times. C, AMPA stimulation after LTP does not change total AMPAR levels. Synaptosomes were treated as in B and then lysed hypotonically and sonicated, followed by [³H]-AMPA binding to determine total AMPAR levels. The decrease in surface AMPARs (B) without changing total AMPAR levels (C) indicates that AMPARs are internalized after AMPA treatment in synaptosomes. D and E, AMPA stimulation leads to the removal of AMPAR from PSDs. Synaptosomes were either left untreated or treated as in B for 10 min. and PSD fractions were purified. Both synaptosome and PSD fractions were immunoblotted with indicated antibodies (D), and O.D. intensities were determined (E). F, AMPA stimulation after LTP does not change NMDAR subunit levels in PSDs. Synaptosome were treated and PSD fractions were prepared as in (D) and immunoblotted with the indicated antibodies. For all experiments, AMPAR levels were determined by measuring specific binding of [³H]-AMPA and values are means±SEM of 3–4 independent experiments. One-way ANOVA on AMPA effect followed by Fisher-Snedecor F test was used in A; F(5,12) = 17.89 ; **, p<0.001 Dunnett's t-test. Two-way ANOVA followed by Bonferroni t-test was used in B; AMPA, F(1,47) = 55.43, p<0.001; Time, F(5,36) = 4.42, p<0.05; AMPA x Time, F(5,47) = 2.413, not significant; ***, p<0.001; ns, not significant. Student's t-test was used in C and E; **, p<0.01; ns, non significant.</p

LTP in synaptosomes does not require new protein synthesis or proteasome-dependent protein degradation.

<p>A, Inhibition of new protein synthesis with cycloheximide does not inhibit LTP in synaptosomes. Synaptosomes were pre-treated either with or without cycloheximide (50 µM) and stimulated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006021#pone-0006021-g002" target="_blank">figure 2A</a>. No significant differences in surface AMPAR levels were found between cycloheximide-treated and control synaptosomes. B, Inhibition proteasome-dependent protein degradation does not inhibit LTP in synaptosomes. Synaptosomes were pre-treated with either the proteasome inhibitor MG-132 (50 µM), lactacystin (10 µM) or control buffer and stimulated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006021#pone-0006021-g002" target="_blank">figure 2A</a>. No significant differences in surface AMPAR levels were found between either proteasome inhibitor and control synaptosomes. In both (A) and (B), surface AMPAR levels were determined by measuring specific binding of [³H]-AMPA to non-permeabilized synaptosomes and values represent means±SEM of 3 independent experiments.</p

Normal Biogenesis and Cycling of Empty Synaptic Vesicles in Dopamine Neurons of Vesicular Monoamine Transporter 2 Knockout Mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity