Figure 1.
Schematic of the prefrontal cortex (PFC) demonstrating location of tissue punches and neurons used for electrophysiology experiments and morphological analysis.
Top: Tissue punches used for western blot analysis centered on the prelimbic (PrL) PFC. Shown are sections of the mouse brain at Bregma 2.00 mm. Bottom: The box shows a bright-field image of a biocytin-labeled layer V pyramidal neuron in the PrL PFC. The inserts show a typical arrangement of the recording and theta-glass stimulation electrode, which was placed within the same layer as the recorded neuron near the basal dendrites.
Figure 2.
Chronic intermittent ethanol exposure (CIE) increases the ratio of NMDA to AMPA currents independent of alterations in synaptic glutamate release.
A) Examples of EPSCs from neurons of a control and CIE-exposed mouse. Left: The NMDA/AMPA ratio was significantly larger in slices from control versus CIE exposed mice. Right: Changes in the amplitude of the NMDAR current was not accompanied by increases in the decay time constant. B) CIE exposure did not affect the frequency or average amplitude of mEPSCs. The top left shows representative traces of pharmacologically isolated mEPSCs in a slice from a control mouse. The right insert shows averaged mEPSCs from a control (black) and CIE (no-withdrawal) mouse, respectively. *p<0.5 significantly different from the vehicle control group (ANOVA and post-hoc analysis using unpaired Student's t-tests). C) Histogram of the amplitude distribution of mEPSCs from all cells over 10 min of recording (control, black; CIE no-withdrawal, blue; CIE 7 days withdrawal, red). The insert shows the same data replotted as cumulative frequency distribution to show that the relative amplitude distribution of synaptic events did also not change.
Figure 3.
Chronic intermittent ethanol (CIE) increases the density of mature dendritic spines in layer V mPFC pyramidal neurons and produces a transient increase in NMDARs.
A) Representative blots of NMDAR and AMPAR subunits in control and CIE exposed mice. B) CIE significantly increased expression of NR1 and NR2B subunits of NMDARs in a PSD-enriched fraction (n = 6–8 mice/group; two-tailed t-tests; *p<0.05) in tissue obtained immediately after the last episode of alcohol exposure. The levels of NR1 and NR2B had returned to baseline after 1- week of withdrawal (WD). C) Representative image of the basal dendrites and dendritic spines of a layer V pyramidal neuron in the mPFC (arrow shows cell body and arrowhead denotes axon). D) Representative images of diolistic labeling of basal dendrites from control and CIE exposed mice. Also shown is automated filament detection and classification of the dendritic shaft (grey) and spines (n = 5–6 mice/group; stubby = red, long = blue, mushroom = green, filopodia = pink). E, G) Total spine density was not altered between control and CIE exposed mice at 0 d or 7 d WD (SAS Proc Mixed model; p>0.05). F, H) The density of mushroom spines was increased by CIE exposure at both time point (SAS Proc Mixed model; *p<0.05).
Figure 4.
Chronic intermittent ethanol (CIE) exposure alters the time course of STDP.
EPSPs were induced by focal stimulation close to basal dendrites of the recorded neuron in layers V/VI. Pairing an EPSP with a burst of postsynaptic action potentials lead to consistent increases in all 3 treatment groups (control, CIE no withdrawal, and CIE followed by 7 days of withdrawal) at 20–30 minutes post pairing. However, at 50–60 minutes post-pairing the EPSP amplitude in CIE exposed mice was significantly increased relative to controls in both the no-withdrawal and one-week withdrawal group. Induction of STDP was NMDAR-dependent: In the presence of the NMDAR antagonist APV the burst of postsynaptic action potentials did not result in a significant change from baseline in slices prepared from control animals. The bar graph shows the relative increase in EPSP amplitude relative to the 10 minutes baseline at 2 different time points (20–30 min and 50–60 min post STDP induction, respectively) for the 4 groups. At 20–30 min, the EPSP amplitude was significantly increased over baseline in all groups. The relative amount of LTP at 50–60 minutes post-pairing was significantly different between control and CIE mice. This was due both to a decrease in potentiation at the late time-point and to the continued increase in LTP in both CIE-exposed groups of mice. *p<0.5 significantly different from baseline (20–30 min) or the vehicle control group (50–60 min), respectively (Repeated measures ANOVA and post-hoc analysis using unpaired Student's t-tests).
Figure 5.
Chronic intermittent ethanol (CIE) exposure reduces the ability of mice to shift attention towards previously unrewarded stimuli in order to learn a new response strategy.
A) CIE does not interfere with the ability to reverse a response discrimination in a T-maze. Mice were trained on a response discrimination (Response) that required them to always turn towards one side to obtain food reward. After 3 cycles of CIE or air exposure and 3 days of withdrawal, mice were retested (Retest) using the same turn discrimination. Animals in both groups showed a small improvement in performance on Retest day, indicating that CIE did not impair the retention of the previously learned strategy. On the following day, mice were required to reverse their strategy (Reversal) and turn towards the opposite arm to obtain the reward. The bar graph in A1 shows the total number of trials to criterion on the 3 test days for mice in the control (n = 8) and CIE (n = 8) groups. Data in A2 shows the number of reinforcers earned (i.e. the number of correct choices) during training (Response and Retest Day). No differences were observed between the treatment groups on any test day. B, C) Attentional Set-shifting is impaired in CIE exposed mice. Mice were trained on a response discrimination task as described in A with the addition of a visual cue (Response). After 3 cycles of CIE or air exposure and 3 or 7 days of withdrawal, mice were retested on the Response Discrimination (Retest). On the following day, mice were trained to shift attention to the visual cue to obtain food reward (Shift to Visual Cue). B1) CIE exposed mice required significantly more trials to reach performance criterion on Reversal Day. B2) Changes on Reversal Day were not due to differences in association strength established during training. The number of reinforcers earned (i.e. the number of correct choices) was not different in all treatment groups. C) Analysis of the types of errors committed on Reversal Day revealed that CIE exposed mice made significantly more perseverative errors than mice in the control group. Other types of errors did not differ significantly, indicating that once the new strategy was acquired, the mice had no difficulty in maintaining the new response strategy. All data are expressed as means ± SEM. **p<0.01 and *p<0.05 significantly different from the vehicle control group (ANOVA and post-hoc analysis using unpaired Student's t-tests).