PMC

AMPAR-mediated mEPSC amplitude, but not conductance was increased in 1-day FZP-withdrawn rats. mEPSC amplitude was increased in 1-day FZP-withdrawn rats without an effect on mEPSC frequency, rise time or decay kinetics, or RMP. (a) Representative plot of the results of non-stationary noise analysis (NSNA) of mEPSCs showed an increase in current amplitude, but no change in slope conductance in an FZP-withdrawn (open circles/dotted line) vs a CON (close circles/solid line) neuron. (b) Mean mEPSC conductance in CA1 neurons from CON (closed bars, n=6) and 1-day FZP-withdrawn (open bars, n=7) rats indicated no significant difference in AMPAR conductance between experimental groups at this time point.

CaMKII subunit levels in CA1 PSD-enriched subcellular fractions as a function of time after drug withdrawal. Representative immunoblots of total CaMKIIα, pThr²⁸⁶-CaMKIIα, and CaMKIIβ protein with their respective GAPDH loading controls are shown in PSD-enriched subfractions of CA1 minislices from (a) 1-day and (b) 2-day FZP-withdrawn rats in the leftmost panels. Histograms of integrated signal density as a percent of the paired control density (n=4–7 lanes/group) are shown to the right. Total-CaMKIIα expression levels were significantly increased in 2-day FZP-withdrawn rats. No significant changes in pThr²⁸⁶-CaMKIIα or CaMKIIβ expression levels were found. Asterisks denote p<0.05.

Greater NAS inhibition in neurons from 1- and 2-day FZP-withdrawn rats. Representative AMPAR-mediated mEPSCs (a, 1 day; d, 2 days) recorded before (solid line) and after (dotted line) external application of the potent spermine analog, NAS (100 μM). The percent NAS inhibition of mEPSC current amplitude was increased (b, 1 day; e, 2 days) in CA1 neurons from FZP-withdrawn rats (p<0.05). Bath application of NAS abolished the increased AMPAR-mediated mEPSC amplitude in FZP-withdrawn neurons after (c) 1 day (n=7) and (f) 2 days (n=12) and was without effect in control neurons (1 day, n=6; 2 days, n=7), supporting the hypothesis that AMPAR current potentiation is mediated by synaptic incorporation of GluA2-lacking AMPARs. Asterisks denote p⩽0.05 or ^**p⩽0.01.

GluA subunit levels in CA1 PSD-enriched subcellular fractions as a function of time after drug withdrawal. PSD-enriched subfractions were collected by ultracentrifugation of TritonX-100-resistant membranes pooled from three hippocampal CA1 minislices as described in Materials and methods. Representative immunoblots of total GluA1 and pSer⁸³¹GluA1 with their respective loading controls are shown in PSD-enriched subfractions from (a) 1-day and (b) 2-day FZP-withdrawn rats in the leftmost panels. Histograms of integrated signal density as a percent of paired control density (n=4–11 lanes/group) are shown to the right. Only total-GluA1 expression levels were significantly enhanced in 1-day FZP-withdrawn rats, whereas both total and pSer⁸³¹GluA1 levels were increased in 2-day FZP-withdrawn rats. There were no changes in GluA2 levels in 1-day (or 2-day; ; ) FZP-withdrawn rats. There were also no changes in GluA3 expression levels in the PSD-enriched fraction from 2-day FZP-withdrawn rats. Asterisks denote p<0.05.

Both AMPAR amplitude and single-channel conductance was reversed by intracellular inclusion of a CaMKII inhibitor. As recorded in ACSF alone () and as shown in , mEPSC (a) amplitude and (c) conductance were significantly increased in 2-day FZP-withdrawn rats in the negative control condition, with scrambled autocamtide inhibitory peptide (sAIP) in the whole-cell micropipette. (b) Representative plot of the results of NSNA of mEPSCs showing an increased current amplitude and slope conductance in an FZP-withdrawn neuron with sAIP (open circles/dotted line) vs a CON neuron with sAIP (close circles/black solid line). Intracellular inclusion of the active CaMKII inhibitor, +AIP reversed both the increased AMPAR-mediated mEPSC (a) amplitude and (c) elevated single-channel conductance to control levels (+sAIP) as illustrated in (b) (gray circles/gray solid line). CaMKIINtide inclusion in a few neurons had a similar effect, suggesting that AMPAR potentiation is mediated by CaMKIIα activation. ^*p<0.05; ^**p<0.01.

The slope of the input–output (I/O) relationship, but not paired-pulse facilitation (PPF) was increased in CA1 neurons from 2-day FZP-withdrawn rats. (a) Evoked EPSC (V_H=−80 mV) amplitude (pA) was plotted as a function of stimulus intensity (V). Linear regression of pooled I/O relationships showed an ∼2.5-fold increase in the stimulus response relationship in CA1 neurons from FZP-withdrawn (open circles, n=9) compared with control (closed circles, n=7) rats. Inset: Representative traces of eEPSCs elicited at 3.6 V (solid line: CON; broken line: FZP). Scale as in inset in (c). (b) The mean slope of the I/O curve derived from the fits of individual I/O curves generated in neurons from FZP-withdrawn rats (440.0±79.4 pA/V, n=9) was also significantly greater than that from control rats (205.3±44.5 pA/V, n=7). The data are consistent with an increase in CA1 neuron AMPAR synaptic efficacy in FZP-withdrawn rats. Asterisks denote p<0.05. (c) PPF of AMPAR was unchanged in 2-day FZP-withdrawn rats. The amplitude of AMPAR-mediated eEPSCs (V_H=−80 mV) after half-maximal stimulation of the Schaffer-collateral pathway. Paired-pulse stimulation was applied and the response was recorded at the interstimulus intervals ranging from 25 to 200 ms in 25 ms increments. Inset: Representative paired EPSC traces recorded in CA1 neurons from control and FZP-withdrawn rats. Paired EPSC amplitudes were calculated as the difference between baseline before the stimulus artifact and EPSC peak. PPF was calculated as (EPSC2−EPSC1)/EPSC1 × 100. Percent facilitation was plotted (CON, closed circles, n=5; FZP: closed circles, n=5) and fit with a single-exponential decay function. No significant differences between groups were found at any interstimulus interval suggesting that glutamate release onto CA1 neurons was unaltered.