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Alteration of sensory input can change the strength of neocortical synapses. Selective activation of a subset of whiskers is sufficient to potentiate layer 4-layer 2/3 excitatory synapses in the mouse somatosensory (barrel) cortex, a process that is NMDAR-dependent. By analyzing the time-course of sensory-induced synaptic change, we have identified three distinct phases for synaptic strengthening in vivo. After an early, NMDAR-dependent phase where selective whisker activation is rapidly translated into increased synaptic strength, we identify a second phase where this potentiation is profoundly reduced by an input-specific, NMDAR-dependent depression. This labile phase is transient, lasting only a few hours, and may require ongoing sensory input for synaptic weakening. Residual synaptic strength is maintained in a third phase, the stabilization phase, which requires mGluR5 signaling. Identification of these three phases will facilitate a molecular dissection of the pathways that regulate synaptic lability and stabilization, and suggest potential approaches to modulate learning.
Neocortical synapses are modified by experience, a process that can occur throughout the lifetime of an animal but is especially pronounced during early postnatal development (Crair & Malenka 1995; Feldman et al 1998; Hensch 2005; Kirkwood et al 1995; Wen & Barth 2011). Because alterations in neocortical circuits are thought to underlie long-term memories (McClelland 1998; Wiltgen et al 2004), the cellular and molecular pathways required for synaptic plasticity in this area have been of great interest. Of critical importance is identifying the specific brain area and set of synapses that have been altered by experience, a criterion that has been perhaps best met by analyses in primary sensory cortex. Here we employ selective whisker activity to investigate the procession of changes in excitatory synaptic strength at layer 4 inputs on layer 2/3 pyramidal cells.
The organization of facial vibrissae is recapitulated in somatotopically precise columns in the barrel cortex, an arrangement that facilitates unequivocal identification of the cortical representation of each whisker (Feldman & Brecht 2005). This feature has been exploited in many in vitro studies to evaluate synaptic plasticity induced by whisker manipulation in vivo (Bender et al 2006; Clem et al 2008; Dachtler et al 2011; Finnerty et al 1999; Hardingham et al 2008; Jacob et al 2012; Jiao et al 2006; Takahashi et al 2003).
Previously we have shown that selective whisker removal (leaving only a single whisker on one side of the snout) selectively strengthens synapses in the spared barrel column within 24 hrs of altered sensory input (Clem & Barth 2006). The rules that govern this plasticity are complex, where NMDARs are required first for the potentiation of spared whisker inputs at layer 4-2/3 synapses, but then mediate synaptic depression (Clem et al 2008). NMDAR-mediated decay of synaptic potentiation has been observed in other systems, such as the hippocampus (Qi et al 2012; Villarreal et al 2002), suggesting that this transition in NMDAR-function might occur across multiple synapse types in the brain.
To understand how experience might persistently alter neocortical synapses, we monitored synaptic potentiation induced by sensory activity at short time intervals. Within a few hours following selective whisker removal, layer 4-2/3 synapses in the spared barrel column grow significantly stronger, reaching their peak amplitude after 12 hours of altered whisker experience. After this initiation phase, sensory input triggers an NMDAR-dependent reduction in synaptic strength, specifically at recently potentiated synapses. This labile phase is transient, lasting ~12 hrs, before synapses are stabilized at a new, larger amplitude compared to control animals.
After the termination of the labile phase, increases in synaptic strength appear to be stabilized, in a process that requires signaling via mGluR5. These multiple phases of synaptic plasticity may be common across many types of excitatory synapses in the CNS, and will provide a platform for analysis of the specific molecular changes that occur during experience-dependent modification of synaptic function. In addition, these results suggest strategies for “memory” modification, using both pharmacological and behavioral approaches to regulate synaptic strength.
Two sensory paradigms were used in this study: single whisker experience (SWE) vs. single row experience (SRE). In the SWE paradigm, bilateral whisker deprivation was performed where all but the D1 whisker on one side of an animal's snout was plucked (Glazewski et al 2007). In the SRE paradigm, all whiskers were deprived bilaterally except a single set of D row whiskers on one side (Finnerty et al 1999). Heterozygous mice from the 1-3 fosGFP line (backcrossed 12-18 generations into the C57Bl6 strain purchased from Harlan) transgenic line (aged P12-P14, of either sex) were used for experiments where SWE was induced. Because no difference in Sr-EPSC amplitudes have been observed between fosGFP+ and fosGFP- layer 2/3 neurons (Clem & Barth 2006), values for all cells were grouped. More than 75% of recorded cells from transgenic mice were fosGFP-. For SRE, almost all experiments (36/40 animals) were performed in wild-type mice. Animals were returned to their home cages for varied amount of time (0-72 hrs) before recording. Control animals were whisker-intact littermates of deprived animals from the same age range (P12-14), and included fosGFP+/- and fosGFP -/- littermates, or were from an in-house C57Bl6 colony established from Harlan. Because control animals did not undergo whisker plucking, the data from these animals are referred to as “0 hr,” to indicate the baseline response, and recordings were not restricted to a single barrel column. Whisker stimulation occurred during the course of normal animal behavior; individual whiskers were not directly stimulated by the experimenter.
Animals were anesthetized with isoflurane and decapitated. Slices were prepared in two different ways according to the type of sensory experience (SWE vs. SRE) that animals underwent in vivo. Coronal slices with 350 μm thickness for SWE-treated animals were vibratome sectioned in artificial cerebrospinal fluid (ACSF) at 2-6°C composed of (mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 26.2 NaHCO3, 11 glucose and equilibrated with 95/5% O2/CO2. Slices from SRE-treated animals were prepared by an “across-row” protocol (Clem et al 2010; Finnerty et al 1999). The dissected brain was put on a flat surface (no incline) and one cut was made at the posterior end of the brain along a 45-degree plane towards the midline (Figure 1A). The hemisphere contralateral to the spared whiskers was saved and the sectioning plane was mounted for slice preparation.
The barrel column representing the spared D1 whisker in SWE-treated animals was identified after at least 18 hr SWE by enhanced fosGFP expression and relative position to the hippocampus in acute brain slices. The spared D row in SRE-treated animals was identified as the fourth barrel from the lateral side of slices that contain 5 barrels (Figure 1B; barrel rows A to E, lateral to medial). Typically, one to two slices per brain contained the entire complement of 5 barrels.
Slices were maintained and whole-cell recordings were performed at room temperature. Somata of lower layer 2/3 pyramidal neurons in barrel cortex (i.e. presumptive layer 3) were targeted for whole-cell recording with borosilicate glass electrodes with a resistance of 4-8 MΩ. Electrode internal solution was composed of (in mM): 130 cesium gluconate, 10 HEPES, 0.5 EGTA, 8 NaCl, 10 Tetraethylammonium chloride (TEA-Cl), 4 Mg-ATP and 0.4 Na-GTP, 5 QX-314 at pH 7.25-7.30, 290-300 mOsm and contained trace amounts of Alexa-568. Pyramidal cell identity was confirmed after the recording session by pyramidal somata morphology and the presence of dendritic spines. Only cells with Rseries ≤ 30 MΩ and Rinput ≥ 200 MΩ, where changes in either measurement were less than 30% were included for analysis. Stimulation of presynaptic afferents at 0.1 Hz was performed using a glass monopolar electrode placed in the center of the barrel in layer 4. Postsynaptic responses from layer 2/3 pyramidal neurons within the same barrel column were recorded. Electrophysiological data were acquired by Multiclamp 700A (Axon Instruments, Foster City, CA) and a National Instruments acquisition interface. The data were filtered at 3 kHz and digitized at 10 kHz and collected by Igor Pro 6.0 (Wavemetrics, Lake Oswego, Oregon). Extracellular simulation was controlled by a Master-8 (A.M.P.I, Israel) and a stimulus isolator Isoflex (A.M.P.I, Israel).
To measure the amplitude of stimulus-evoked mEPSCs, Sr++ (3 mM) was substituted for Ca++ in ACSF to drive asynchronous glutamate release. D-APV (50 μM) and picrotoxin (Ptx, 50 μM) were included to pharmacologically isolate AMPAR-mediated EPSCs. Layer 2/3 pyramidal neurons were voltage-clamped at -70 mV. The evoked response has an initial synchronous component (~50 ms post the stimulus artifact) which was excluded in the analysis. Isolated, asynchronous events that occurred from 50-500 ms after the stimulus were manually selected and analyzed using Minianalysis software (Synaptosoft, Inc. Decatur, GA). The detection threshold for events was set at 2× RMS noise (usually around 4-5 pA) and data were filtered with a low-pass filter at 1 kHz.
The frequency and amplitude of all EPSCs onto layer 2/3 neurons were collected using 0.5 μM TTX (Sigma) in bath to block all firing activity in the slice. In this case, mEPSC amplitude measurements cannot be attributed to a single pathway, and represent the sum of diverse inputs to the cell.
The NMDAR antagonist, CPP (10 mg/kg body weight) or the mGluR5 antagonist, MPEP (10 mg/kg) was injected intraperitoneally at different time points, and whole-cell recordings were performed on injected animals 24, 48 or 72 hours after the onset of SWE. Injections were administered once, and animals were returned to their home cages until sacrifice. In some SWE-treated animals, CPP was injected in combination with the removal of the remaining whisker, where CPP injection was performed immediately after the remaining whisker was removed.
Approximately 100 randomly-chosen Sr-EPSC events were selected and then grouped and averaged to obtain the average EPSC for each cell. Averaged Sr-EPSC traces for each experimental condition were obtained by first averaging responses from individual cells and then averaged these traces across all cells in the group. Mean Sr-EPSC amplitudes were averaged across cells for each condition. In general, control data were collected in interleaved experiments for each specific research question that was addressed.
Statistical significance was calculated between groups for each timepoint or drug manipulation. To accommodate random effects caused by animal-to-animal variability in our datasets, we employed a generalized linear mixed model followed by simultaneous multiple pair-wise comparisons. P values were adjusted for multiple comparisons described by Benjamini and Hochberg (Benjamini & Hochberg 1995). Cumulative distributions of EPSC event amplitude for different conditions were compared using Kolmogorov-Smirnov Test (KS test). All statistical analyses were performed using the software program R. A p-value less than 0.05 was considered statistically significant, where * and ** indicate p≤0.05 and 0.01, respectively.
Previous experiments to examine whisker-induced synaptic strengthening have relied upon expression of the immediate-early gene c-fos in the fosGFP transgenic mouse to identify the barrel column corresponding to the spared whisker (Barth et al 2004). However, this method cannot reliably identify the spared barrel column at time intervals less than ~18 hrs of single-whisker experience (SWE) due to poor resolution of the fosGFP signal in the spared versus the deprived barrel columns. To examine the early timecourse of plasticity, another form of selective whisker activation was adopted that preserved not one, but all whiskers in the D- row (single-row experience; SRE; (Finnerty et al 1999)). This method allows unambiguous identification of the spared whisker representation in wild-type mice, in acute brain slices prepared to preserve the row identity (Figure 1).
The effects of SRE on excitatory synaptic strength at individual layer 4-2/3 contacts can be assessed using a Sr-replaced ACSF solution to desynchronize neurotransmitter release, (Goda & Stevens 1994; Xu-Friedman & Regehr 1999). This method specifically evaluates post-synaptic changes in synaptic strength, and normalizes stimulation conditions across different brain slices and animals. In undeprived, control animals (0 hr SRE), Sr-EPSC amplitudes were identical between SWE and SRE (Figure 1C-E; SWE 0 hr 9.9±0.80 pA, vs. SRE 0 hr 9.2±0.37 pA, p=0.3), suggesting that there is no bias in the population of synapses under investigation that might be introduced by different slicing protocols. The magnitude of synaptic potentiation after SWE was slightly lower than SRE (Figure 1E; SWE24hr 11.9±-0.43 pA, vs. SRE24hr 10.7±0.40 pA, p=0.1), likely because the retention of a single whisker more strongly drives plasticity in layer 4-2/3 circuits. However, in both cases, a significant increase in mean Sr-EPSC amplitude was observed after 24 hrs.
These initial comparisons suggest that SWE and SRE are similar in terms of their capacity to increase synaptic strength. Thus, SRE enables analysis of the early time points of sensory experience and can be used as a tool to study the progression of synaptic potentiation in vivo.
Given that there was an overall smaller increase in synaptic strength after SRE compared to SWE at 24 hrs, it was unclear whether smaller increments of change could be resolved using this assay. However, this concern was unwarranted, as shorter intervals of SRE were in fact more effective at driving robust increase in Sr-EPSC amplitude.
Compared to undeprived, control animals, mean Sr-EPSC amplitude at 6 and 12 hrs showed a progressive, linear increase (Figure 2B,C; 0 hr 9.2±0.37 pA; 6 hr 11.4±0.55 pA; 12 hr 13.1±0.40 pA, 0 vs 6 hr, p=0.03; 6 vs. 12 hr, p=0.08; 0 vs. 12 hr, p<0.0001). Overall, Sr-EPSC amplitude increased by about 40% from 0 hr to 12 hr and the rate of synaptic strengthening was relatively constant between 0-6 hr and 6-12 hr (~0.3 pA/hr). Although individual events varied over a large amplitude range (3.2-48.7 pA), a cumulative distribution of event amplitudes shows a progressive rightward shift from 0 hr to 6 hr and from 6 hr to 12 hr (Figure 2D; 0 vs. 6 hr, p<0.001; 6 vs. 12 hr, p<0.001). These data show that during the first 12 hrs of SRE, excitatory synaptic strength can be profoundly enhanced, and that the magnitude of potentiation has been underestimated by previous analyses that focused on later timepoints.
The fact that Sr-EPSC amplitudes were significantly larger at 12 hrs than at 24 hrs suggested that further experience might not just be ineffective at inducing further potentiation, but might be actively suppressing existing gains. Sr-EPSCs recorded at 24 hr of SRE were ~2 pA smaller compared to the peak measured at 12 hr (Figure 3A,B; 18 hr 12.4±0.56 pA and 24 hr 10.7±0.35 pA, 12 vs. 18 hr, p=0.5, 18 vs. 24 hr p=0.09, 12 vs. 24 hr, p=0.03). This decrease in synaptic strength was also evident from an overall leftward shift in the cumulative distribution (Figure 3C; 12 vs. 18 hr, p<0.001; 18 vs. 24 hr, p<0.001), where all events appeared to be reduced, compared to an increase in the frequency of very small events that might drive down the mean amplitude. The calculated rate of synaptic weakening between 12 and 24 hr of SRE was ~0.2 pA/hr, slightly smaller than that of synaptic strengthening between 0 and 12 hr, resulting in a net gain in input amplitude at 24 hr.
Since the spared barrel could be identified in SWE-treated fosGFP mice at 18 hr after the SWE-onset, the effects of SRE and SWE could be directly compared at this timepoint. Consistent with the results from SRE-treated animals at 18 hr, mean Sr-EPSC amplitude was higher at 18 hr compared with 24 hr in SWE-treated animals (Figure 3D). There was no significant difference in the fold-change in synaptic strength at 18 hr or 24 hr between the two conditions (Figure 3D). Thus, both whisker deprivation paradigms showed an early potentiation phase where synaptic strength is significantly enhanced and a labile phase, where synaptic strength is then reduced. These data suggest there is a transition in how layer 4-2/3 synapses respond to selective whisker activation, where long periods of selective whisker stimulation activity do not progressively increase synaptic strength.
Synaptic scaling, or the reduction of synaptic weights based upon increased network activity, has been observed in neocortical neurons in young postnatal animals (Turrigiano et al 1998). Synaptic scaling is a slow process that occurs across all excitatory inputs to the cell, requires NMDAR-activation, and typically requires several days to become manifest (Turrigiano et al 1998) but see (Sutton et al 2004). It is conceivable that the synaptic weakening observed during the labile phase might be concurrent with a delayed-onset cell-wide synaptic rescaling. Thus, the initiation phase would represent an input-specific synaptic strengthening, and the subsequent labile phase would be a global rescaling of synaptic weights in order to maintain total input strength over some target range.
To examine the mean amplitude for all excitatory inputs onto the cell, we measured mEPSCs for layer 2/3 neurons in the presence of the Na-channel blocker, TTX (Figure 4A). Mean mEPSC amplitude for layer 2/3 neurons from control undeprived animals was 10.5±0.5 pA (Figure 4B-C,E; age range P13-14). This value was compared to mean mEPSC amplitude during the middle of the labile phase, at 18 hrs of SRE, as we reasoned that cell-wide synaptic scaling might be most pronounced here. However, mean mEPSC values were unaltered at this timepoint (Figure 4B-C,E; 10.3±0.4 pA; age range P13; vs. control 10.5±0.5 pA, p=0.8). The cumulative distribution of event amplitude for these two data points were overlapping (Figure 4D), however, there was a statistically significant difference (p=0.04). This should be compared to the shift in Sr-EPSC amplitude event distribution at the layer 4-2/3 pathway for these two timepoints (Figure 3C, 0 hr vs. 18 hr, p<0.001). The frequency of mEPSCs between the control and 18 hrs SRE-treated animals was not significantly altered (Figure 4F, p=0.6).
The identification of the specific time interval where gains in synaptic strength can be reduced or reversed facilitates identification of cellular mechanisms that underlie this form of synaptic metaplasticity. Previously we found that after the onset of whisker-driven potentiation, subsequent NMDAR-activation triggers synaptic depression (Clem et al 2008; Wen & Barth 2012). Using the time intervals identified above, we investigated how systemic NMDAR-blockade administered at different phases of plasticity could influence synaptic strength (Figure 5). Intraperitoneal (i.p.) injections of the competitive NMDAR-antagonist CPP were carried out at various timepoints, and mean Sr-EPSC amplitude was calculated 24 hrs after whisker removal. Since all analysis was carried out 24 hrs after whisker removal (when the spared barrel column was visible by fosGFP expression in an acute brain slice), the SWE preparation was employed in these experiments.
Our previous work showed that injection of NMDAR-antagonist at the onset of SWE was sufficient to prevent experience-dependent synaptic strengthening, consistent with a requirement for NMDARs in long-term potentiation at layer 4-2/3 synapses in vitro (Clem et al 2008). Here we show that slightly later injections, 6 hrs after the onset of SWE, can also block increases in layer 4-2/3 Sr-EPSC amplitude (Figure 5A and D; SWE6hrCPP 10.59±0.37 pA vs. control 9.87±0.24 pA, p=0.4), suggesting that NMDAR-activation at this time is still required for synaptic strengthening at these synapses.
Mean Sr-EPSC amplitude in the spared barrel column peaks at 12 hrs after the onset of whisker manipulation, a putative transition point from the initiation to the labile phase. If NMDARs are required for synaptic weakening during the labile phase, blockade of these receptors could preserve these early gains in Sr-EPSC amplitude. This was indeed the case. CPP injection at 12 hrs after SWE onset prevented the expected decline in Sr-EPSC amplitude at 24 hrs (Figure 5B and D; SWE12hrCPP 15.72±1.05 pA vs. SWE24hr 12.01±0.63 pA, p=0.002). These data indicate that that the transition from NMDAR-potentiation to NMDAR-depression has occurred by this timepoint, and suggest that blockade of NMDARs is sufficient for maintenance of the early gains in synaptic strength induced during the potentiation phase.
To test whether input-specific mechanisms that rely on ongoing whisker activity were important for synaptic lability, the remaining whisker was removed from SWE animals, 18 hrs after onset. The whisker was removed by plucking the hair from the follicle so that not even the whisker stub remained (Figure 6A).
Interestingly, whisker removal during the labile phase appeared to enhance Sr-EPSC amplitudes compared to values obtained after 24 hrs of SWE (Figure 6B,C; SWE24hr 11.89±0.23 pA vs. SWE18hrpluck 13.86±0.65 pA, p=0.3). Indeed, mean response amplitudes from animals plucked at 18 hrs and assessed at 24 hrs were identical to values following 18 hrs of SWE (Figure 6B,C; SWE18hrpluck 13.86±0.65 pA vs. SWE18hr 13.92±1.38 pA, p=0.8), suggesting that prior gains in synaptic strength were fully maintained. Consistent with this, the cumulative distribution of Sr-EPSC amplitudes was right-shifted in plucked animals (Figure 6D; p<0.0001). These data show that ongoing sensory-driven activity may reverse prior plasticity during the labile phase. Notably, trimming the spared whisker (leaving a small stub on the snout) at the same 18 hr timepoint did not preserve synaptic potentiation (SWE24hr 11.55±0.50 pA, n=18 cells vs. SWE18hrTrim 11.9±0.69 pA, n=10 cells, p=0.7). This result suggests that input via the whisker stub may be sufficient to engage synaptic depression during the labile phase.
The finding that whisker removal, like NMDAR-blockade, is sufficient to maintain prior gains in Sr-EPSC suggested that these two manipulations might be related. To test this, we examined the effect of simultaneous whisker removal and NDMAR-blockade (Figure 6E). If whisker removal and in vivo NMDAR-blockade acted through two different pathways to increase Sr-EPSC amplitude, we might observe an additive effect of these two manipulations together. Alternatively, if these mechanisms were part of the same pathway, there should be no further increase.
At 18 hrs after SWE onset, animals were injected with CPP and the remaining spared whisker was removed (SWE18hrpluck+CPP). Sr-EPSC amplitudes at layer 4-2/3 inputs were then evaluated 6 hrs later (Figure 6E). CPP injection had no greater effect when combined with whisker removal at 18 hrs (Figure 6F,G; SWE18hrpluck 13.86±0.65 pA vs. SWE18hrCPP 13.35±0.47 pA vs. SWE18hrpluck+CPP 13.19±0.74 pA, p=0.9 for all comparisons). The cumulative distribution of Sr-EPSC amplitudes was also not different between the three cases (Figure 6H). Thus, we propose that loss of sensory input during the labile phase can act to maintain prior gains in synaptic strength by circumventing NMDAR-dependent depression.
Given the opposition between NMDAR-dependent potentiation and NMDAR-dependent depression on EPSC amplitude after the onset of synaptic strengthening in vivo, what happens during longer periods of selective whisker experience? Sr-EPSC amplitudes at layer 4-2/3 synapses in the spared whisker barrel column remain elevated for days after the onset of SWE (Figure 7; SWE48hr 11.30±0.56 pA vs. SWE72hr 11.13±0.39 pA, compared to SWE24hr 11.75±0.44 pA; Figure 1). These data indicate that NMDAR-dependent depression is not sufficient to eliminate experience-dependent synaptic potentiation over these longer time periods. This might be the case because the labile phase is transient, or because the rate of depression progressively slows.
To examine whether NMDAR-dependent synaptic weakening was still active at these later timepoints, CPP was injected at 36 hrs after SWE onset, and Sr-EPSCs were evaluated at 48 hrs (Figure 7A). If the labile phase is prolonged, we expected that this 12 hr treatment would result in larger Sr-EPSCs, as was observed with earlier injections (Figure 5). However, we found that later NMDAR-blockade had no effect on mean Sr-EPSC amplitude (Figure 7A,C; SWE48hr 11.30±0.56 pA vs. SWE36hrCPP 11.14±0.73 pA, p=0.8). Later injections of CPP, 60 hrs after SWE onset, also showed no effect at 72 hrs (Figure 7B,C; SWE72hr 11.13±0.39 pA vs. SWE60hrCPP 11.85±0.50 pA, p=0.8). Unlike NMDAR-blockade at 12-18 hrs following SWE onset, later blockade has no effect on excitatory synaptic strength. Thus, NMDAR-activation does not reduce experience-dependent synaptic strengthening in the spared barrel column after the first 36 hours of selective whisker experience, where responses appear to be stabilized.
Previous work suggested that group I metabotropic glutamate receptors (mGluRs) might be required to maintain synaptic strength during the period we have now identified as the labile phase (Clem et al 2008). Here we identify that mGluR5 is the specific group I mGluR required, using the mGluR5-specific antagonist MPEP. I.p. injection of MPEP during the labile phase (18 hrs post-SWE onset) was sufficient to eliminate whisker-induced potentiation, reducing Sr-EPSC amplitudes back to baseline levels (Figure 8A,B; SWE18hrMPEP 9.94±0.65 pA vs. SWE24hr 12.72±0.45 pA, p=0.0002). These results extend findings reported earlier for the non-specific group I antagonist, AIDA (Clem et al 2008), identifying mGluR5 as the critical receptor subtype involved in this process.
Is mGluR5 activity required to offset the depressing function of NMDAR-activation only during the labile phase? To answer this question, animals were injected with MPEP 36 hrs after SWE-onset, and assessed at 48 hrs. This treatment was sufficient to reduce Sr-EPSC amplitudes back to baseline levels (Figure 8C,D; SWE36hrMPEP 9.40±0.45 pA vs. SWE48hr 11.30±0.56 pA, p=0.005; SWE36hrMPEP vs. control, p=0.9). Thus, the persistence of experience-dependent increases in layer 4-2/3 excitatory synaptic strength requires continued activation of mGluR5 signaling pathways.
Do other neocortical synapses that are modified during SWE show the same phases of plasticity? Previous work has shown that SWE leads to the strengthening of layer 2/3-2/3 synapses within the spared barrel column (Wen & Barth 2011). To determine whether these synapses also showed three distinct phases of synaptic plasticity, we examined Sr-EPSC amplitude using a stimulating electrode positioned within the spared column (Figure 9A), at a variety of timepoints following SRE.
In comparison to layer 4-2/3 inputs, synaptic strength appeared to peak earlier for layer 2/3-2/3 inputs, at ~6 hrs following SRE onset (Figure 9B,C; control 8.5±0.4 pA vs. SRE6hr 10.9±0.5 pA, p=0.0006). By 12 hrs, there was a small but significant dip in mean Sr-EPSC amplitude (9.4±0.4 pA, p=0.02 vs. 6 hrs). Synaptic strength appeared to stabilize between 18 and 24 hrs (Figure 9B,C). These data suggest that other synapses within the spared barrel column might undergo a labile phase following potentiation, although the specific timing of these phases might differ.
Here we use sensory experience to drive changes in neocortical circuits in order to investigate the timing and regulation of synaptic potentiation in vivo. The method employed here specifically isolates changes in the post-synaptic response of pathway-specific inputs to layer 2/3 pyramidal neurons. We find that synaptic strengthening at layer 4-2/3 synapses within the spared barrel column(s) proceeds at a constant rate over the first 12 hrs, resulting in a ~40% increase in the amplitude of Sr-EPSCs compared to control, undeprived animals. This early peak is followed by a labile period lasting less than 24 hrs, where ongoing sensory input reduces EPSC amplitude in an NMDAR-dependent manner. Finally, experience-dependent changes are consolidated during the stabilization phase, where NMDAR-activation does not enhance or suppress further changes, but mGluR5 activation is required to maintain prior gains in synaptic strength.
Synaptic neurophysiologists have long postulated that prior synaptic strengthening can alter the requirements for future synaptic plasticity. The relationship between input activity and synaptic change has been formalized by Bienenstock, Cooper, and Munro (Abraham & Tate 1997; Benuskova et al 1994; Bienenstock et al 1982), and abundant experimental evidence has shown that excitatory synapses will depress with low frequency stimulation and potentiate with high frequency stimulation (Dudek & Bear 1992; Kirkwood et al 1993; Malenka & Bear 2004). Furthermore, the relationship between stimulation frequency and synaptic changes can be left-shifted by prior experience (Bear 1995; Philpot et al 2007; Philpot et al 2003). Our data fit nicely into this general model, where a rightward shift of the frequency-response function would lead to depression at recently strengthened layer 4-2/3 synapses. However, our data indicate that this shift may be a temporary phenomenon, identifying a short temporal window of synaptic lability after potentiation has occurred.
The reversibility of experience-induced increase in synaptic strength has parallels in vitro, where subsequent activity following an initial LTP induction can degrade synaptic strengthening, a process that has been referred to as depotentiation (Barrionuevo et al 1980; Bashir & Collingridge 1994; Fujii et al 1991). We have also observed an NMDAR-dependent variant of this process at SWE-potentiated layer 4-2/3 synapses in vitro (Wen & Barth 2012). This process of active decay has also been observed in vivo (Qi et al 2012; Villarreal et al 2002; Whitlock et al 2006; Xu et al 1998; Zhou et al 2003). Our analysis of the timecourse of plasticity at layer 2/3-2/3 suggests that a similar phenomenon might also be occurring at these inputs, albeit with slightly different timing. Thus, the phases we identify here may be observed at many different types of synapses across the nervous system.
Is synaptic downscaling, as a response to increased firing activity in the spared barrel columns, sufficient to account for the labile phase? Prior studies have shown that increasing activity can downscale global EPSC amplitudes in neocortical neurons (Turrigiano et al 1998), and that this homeostatic process requires several days to occur. Although mean mEPSC amplitudes were unchanged between control animals and the middle of the labile phase, our data indicate that there may be some small degree of downscaling, specifically of larger EPSCs, at this time (i.e. Figure 4D). If a cell-wide synaptic downscaling were the best explanation for the decrease in excitatory synaptic strength, it should be manifested similarly for all inputs to the cell as for layer 4-2/3 synapses. This was not the case. We note that previous manipulations to examine homeostatic changes in synaptic strength have required profound modulation of neural activity, such as total blockade of fast GABAergic synaptic transmission (O'Brien et al 1998; Turrigiano et al 1998) or complete sensory deprivation (Desai et al 2002). The lack of strong evidence for synaptic scaling may be related to the relatively modest change in activity introduced by SWE or SRE.
Can synaptic homeostasis occur in a pathway-specific manner? Previous studies suggest that this can occur, although the timescale of this response may be quite different (Hou et al 2011). Indeed, we suggest that this synapse-specific process may be akin to the labile phase identified in the current study. We propose that cell-wide changes in input strength should be considered homeostatic, as the cell globally responds to changes in firing activity, and pathway-specific changes may be part of a mechanistically distinct process. Future experiments will be required in order to differentiate these phenomena.
Our experiments indicate that there is a distinct transition in NMDARs between the initiation and the labile phases. How is NMDAR function altered to reverse the sign of plasticity at layer 4-2/3 synapses? NMDAR activation can strongly influence network activity and change evoked firing rates, indirectly influencing plasticity induction. However, in vivo recordings from anaesthetized mice indicate that mean firing rates of layer 4 neurons are not altered 24 hrs after SWE onset (Benedetti et al 2009). Our previous experiments (Clem et al 2008) show that even when presynaptic activity is controlled and post-synaptic firing is blocked, the sign of plasticity is still inverted in acute brain slices from SWE-treated animals (i.e., LTP-inducing stimuli trigger LTD after 24 hrs SWE), and that this phenomenon depends upon NMDAR-activation and an increase in post-synaptic Ca++. In light of this, we propose that the changes in NMDAR-function are likely to occur at synapses, influencing signaling pathways locally.
Both pre- and postsynaptic NMDARs have been described at layer 4-2/3 synapses (Brasier & Feldman 2008; Rodriguez-Moreno & Paulsen 2008), and our data do not enable us to assess the relative contribution of these two different NMDAR pools. However, we note that our measurements of synaptic strength exclusively measure post-synaptic response, and the overall outcome of this change in NMDAR function during SWE is clearly manifested as a change in post-synaptic AMPAR-mediated currents (Clem & Barth 2006). It will be of interest to examine experience-dependent changes in presynaptic properties at layer 4-2/3 synapses. Identification of these distinct phases of plasticity will facilitate more detailed investigations into how NMDAR function can be altered by prior synaptic strengthening.
Data presented here suggest interventions by which synaptic strengthening – or learning – might be enhanced. It is difficult to identify the exact timing of glutamate receptor blockade required for effects described here (because the specific bioavailability of the injected antagonist was not determined), although prior work indicates that both MPEP and CPP can antagonize mGluR5 and NMDARs for hours after injection, with the mGluR-antagonist effects of MPEP lasting at least 1-2 hours and the NMDAR-antagonist effects of CPP lasting at least 3 hours and up to 24 hrs after injection (Anderson et al 2003; Villarreal et al 2002). In almost all cases, we found a positive effect of antagonist injection, indicating that at a given time range the compound was sufficient to disrupt plasticity processes. It will be of interest to determine the precise time of transition in glutamate receptor function during these three phases of plasticity; this may be facilitated by experiments in acute brain slices (see for example, (Clem et al 2008)).
We found that eliminating input activity (by removal of the spared whisker) phenocopies pharmacological inactivation of NMDARs. Altering input activity is a more accessible approach to enhance stimulus encoding in vivo, compared to systemic administration of NMDAR-antagonists which would have many side-effects. In addition, these findings suggest strategies for memory weakening, by timing input reactivation during the labile phase. Interestingly, this behavioral approach has been used to reduce fear memories in rodents (Monfils et al 2009; Myers et al 2006) and may be useful for treating PTSD in humans (Mahan & Ressler 2012).
Our identification of the initiation, labile and stabilization phases in the plasticity of neocortical synapses has remarkable parallels in human memory studies. Many forms of memory exhibit an encoding, or initiation phase, followed by a labile phase where the “memory trace” is susceptible to interference by presentation of distractor stimuli or of pharmacological compounds that interfere with gene transcription and translation reviewed by (Nader et al 2000). In addition, consolidation –the post-acquisition stabilization of memory – has been well-described in many experimental paradigms (Dudai 2004).
Whisker-induced plasticity in the developing somatosensory cortex may appear distantly related to memory as applied to other human and animal studies. However, it is striking that the distinct phases identified in the current study – which focused on a single class of neocortical synapses – show such strong parallels with psychological phenomenon that describe well-known aspects of memory processes. The anatomical and temporal precision of the current study suggests strategies to modulate memory formation and erasure, and will facilitate molecular investigations into the pathways that underlie transitions in synaptic properties during learning and memory.
This work was supported by a grant from the National Institutes of Health, DA-0171-88. Special thanks to Joanne Steinmiller for expert animal care, and members of the Barth lab for helpful discussions and comments on the manuscript. Thanks also to Bronwyn Woods and Linqiao Zhao for help with application of the generalized linear mixed model for statistical analysis between animal groups.
A patent is pending on the fosGFP transgenic mice.