The cortical whisker representation of the rodent is an excellent system for examining sensory-induced plasticity (Feldman and Brecht, 2005
). In particular, partial whisker deprivation triggers competitive plasticity resulting in greater excitation of cortical neurons in response to spared whisker deflection (Glazewski and Fox, 1996
; Glazewski et al., 2007
). A brief period of single-whisker experience increases the efficacy of AMPAR-transmission in the cortical layer 4-2/3 pathway and results in the appearance of CP-AMPARs at these same inputs (Clem and Barth, 2006
; Clem et al., 2008
In order to target sensory-stimulated neurons with greater ease, we adapted the single-whisker protocol to produce a complete row of spared whiskers. Prior studies in which multiple whisker rows have been spared have demonstrated LTP-like enhancements in transmission within spared whisker columns (Finnerty et al., 1999
; Cheetham et al., 2007
), suggesting that multiple versus single-whisker sparing might yield similar plasticity. Accordingly, mice were deprived of all but one unilateral row of whiskers for a period of 48 hours ().
Figure 1 Single-row experience drives PICK1-dependent increase in AMPAR-EPSC rectification at layer 4-2/3 synapses. (A) Plasticity was induced in PICK1 knockout mice by single-row experience, which entailed the removal of all but a unilateral D row of whiskers (more ...)
Taking advantage of an oblique slice preparation containing an ordered arrangement of the large whisker barrels (Finnerty et al., 1999
), we then examined layer 4-2/3 excitatory synaptic responses to test for the presence of rectifying CP-AMPARs (). In agreement with previous experiments using a single-whisker protocol (Clem and Barth, 2006
), we found that single-row experience triggered an increase in rectification of AMPAR-EPSCs at sensory spared layer 4-2/3 inputs in PICK1 (+/+) animals, but not at deprived inputs within the same slice (). By quantifying the amount of rectification from these I–V plots, we found that significantly more rectification was present in spared synapses from PICK1 (+/+) after single-row experience (, rectification index = control 0.98 ± 0.05 (n = 7), spared 1.41 ± 0.06 (n = 9), deprived 1.01 ± 0.03 (n = 7). In contrast, single-row experience produced no change in AMPAR-EPSC rectification in PICK1 (−/−) mice (, rectification index = control 1.09 ± 0.10 (n = 6), spared 1.04 ± 0.06 (n = 11), deprived 1.00 ± 0.03 (n = 6)), indicating that PICK1 knockouts are defective in CARP. The impairment in PICK1 (−/−) mice could not be overcome by lengthening the period of single-row experience to 72 hrs (72 hrs single-row experience, rectification index = spared 1.00 ± 0.08 (n = 6)), suggesting that the failure to incorporate CP-AMPARs could not be explained by a higher induction threshold.
Having established that PICK1 deletion disrupts CARP, we then asked whether synaptic strengthening was likewise defective in PICK1 (−/−) mice. A commonly-employed indicator of prior synaptic strengthening is the ratio of AMPAR-mediated to NMDAR-mediated current (AMPA:NMDA ratio), in which increases in this ratio reflect augmented AMPAR currents. Surprisingly, recordings performed after single-row experience revealed that AMPA:NMDA ratio was enhanced in both PICK1 (+/+) and PICK1 (−/−) mice within spared barrel columns (, AMPA:NMDA ratio: PICK1 (+/+) = control 0.99 ± 0.07 (n = 16), spared 1.50 ± 0.12 (n = 13), deprived 0.98 ± 0.12 (n = 11); PICK1 (−/−) = control 0.92 ± 0.08 (n = 12), spared 1.36 ± 0.09 (n = 19), deprived 0.98 ± 0.07 (n = 15)). Therefore, although PICK1 (−/−) synapses were lacking in CP-AMPARs, AMPAR-EPSCs were nevertheless potentiated by prior experience.
Sensory-driven enhancement of AMPA:NMDA ratio does not require PICK1. (A) Overlay of EPSCs evoked at −70, 0, and +40 mV. (B) Mean AMPA:NMDA ratio for EPSCs. * p < 0.001 by ANOVA followed by Bonferroni post-hoc comparison.
In order to exclude the possibility that changes in AMPA:NMDA ratio were due entirely to plasticity of NMDAR currents, we examined the amplitude of AMPAR-EPSCs resulting from quantal evoked glutamate release (Sr2+-mEPSCs) by replacing extracellular Ca2+ with Sr2+ (3 mM). Comparison of Sr2+-mEPSCs revealed that amplitudes were significantly increased by single-row experience in both PICK1 (+/+) and PICK1 (−/−) mice (, Sr2+-mEPSC amplitude: PICK1 (+/+) = control 8.67 ± 0.29 (n = 12), spared 11.84 ± 0.50 (n = 10), deprived 9.65 ± 0.29 (n = 10); PICK1 (−/−) = control 9.03 ± 0.22 (n = 8), spared 11.52 ± 0.52 (n = 10), deprived 8.66 ± 0.36 (n = 9)), suggesting that AMPAR plasticity formed the basis for an increase in AMPA:NMDA ratio. Consistent with PICK1-dependent insertion of CP-AMPARs, Sr2+-mEPSCs exhibited faster decay kinetics in spared neurons following single-row experience in PICK1 (+/+) but not in PICK1 (−/−) mice (, Sr2+-mEPSC tau decay: PICK1 (+/+) = control 5.47 ± 0.40 (n = 11), spared 3.77 ± 0.27 (n = 11), deprived 5.51 ± 0.31 (n = 10); PICK1 (−/−) = control 6.46 ± 0.56 (n = 8), spared 5.63 ± 0.39 (n = 10), deprived 6.14 ± 0.41 (n = 9)).
Figure 3 Quantal AMPAR-EPSCs are potentiated by single-row experience independent of PICK1 or CP-AMPARs. (A) Mean quantal AMPAR miniature EPSCs evoked by stimulation in the presence of Sr2+ (Sr2+-mEPSCs) at layer 4-2/3 inputs. (B) Mean amplitude of Sr2+-mEPSCs. (more ...)
Examination of spontaneous mEPSCs from control mice revealed that amplitude, frequency, and decay kinetics of mEPSCs were unaffected by PICK1 genotype (PICK1 (+/+): mEPSC amplitude = 7.49 ± 0.32 pA (n = 7), mEPSC tau decay = 5.26 ± 0.27 ms (n = 7), mEPSC inter-event interval = 170.2 ± 18.6 ms (n = 6); PICK1 (−/−): mEPSC amplitude = 7.50 ± 0.42 pA (n = 7), mEPSC tau decay = 5.18 ± 0.19 ms (n = 7), mEPSC inter-event interval = 152.4 ± 9.9 ms (n = 7)), indicating that PICK1 does not affect synaptic efficacy or synaptic AMPAR phenotype under basal conditions. Thus, while PICK1 promotes CARP, this phenomenon does not occur during normal sensory activity, and is not required for normal development of synaptic responses.
One mechanism by which PICK1 may lead to the appearance of CP-AMPARs at synapses is by altering the distribution of CP-AMPARs between intracellular and surface compartments. Therefore, to determine whether PICK1 affects the localization of AMPAR subtypes, we performed surface biotinylation of barrel cortex tissue. Since the vast majority of forebrain AMPARs likely consist of GluR1 or GluR2, alone or in combination (Wenthold et al., 1996
; Lu et al., 2009
), we focused our attention on these subunits. As predicted, PICK1 knockouts showed higher surface/total levels of GluR2 (, 124.7 ± 6.1% of wildtype (n = 5 animals each)), indicating that PICK1 negatively regulates membrane expression of GluR2-containing AMPARs. Interestingly, PICK1 deletion also resulted in lower surface expression of GluR1 (, 78.8 ± 6.9% of wildtype (n = 4 animals each)). No significant differences in total AMPAR expression levels were observed (, PICK1 (−/−): GluR1 110.6 ± 7.0% of wildtype (n = 4 animals each); PICK1 (−/−): GluR2 92.2 ± 6.9% of wildtype (n = 5 animals each)).
Figure 4 PICK1 facilitates the surface expression of GluR2-lacking AMPARs. (A) Example experiment in which acute barrel cortex slices from PICK1 (−/−) mice and their PICK1 (+/+) littermates were subjected to surface biotinylation assay. The relative (more ...)
In order to directly examine whether PICK1 promotes the surface expression of CP-AMPARs, we employed immunodepletion of GluR2-containing AMPARs from cortical lysates (; as done by Wenthold et al., 1996
; Conrad et al., 2008
) to examine the fraction of GluR1 remaining. Following GluR2 depletion to 1.83 ± 1.16% of input level in PICK1 knockouts and 2.75 ± 0.75% in wildtype littermates (n=7 slices from 5 animals, each genotype), a substantial amount of total GluR1 remained (). However, PICK1 genotype did not significantly affect the fraction of total unbound GluR1 (, PICK1 (+/+) = 20.4 ± 5.8%, PICK1 (−/−) = 15.5 ± 4.6%), suggesting that the total level of CP-AMPARs was similar in PICK1 knockouts. In contrast, the fraction of surface biotinylated GluR1 that remained was significantly lower in PICK1 knockouts (, surface unbound GluR1: PICK1 (+/+) = 4.23 ± 0.4%, PICK1 (−/−) = 2.63 ± 0.4%). This could not be explained by uneven depletion of GluR2 from PICK1 (−/−) lysates, since surface unbound GluR2 did not differ from wildtype littermates (, surface unbound GluR2: PICK1 (+/+) = 0.42 ± 0.2%, PICK1 (−/−) = 0.41 ± 0.1%). Importantly, because our whole-cell recordings do not indicate the presence of CP-AMPARs at synapses under basal conditions, these data suggest that PICK1 facilitates the surface expression of CP-AMPARs at extrasynaptic sites.