1. Matrix Metalloproteinase-9 and Synaptic Plasticity in the Central Amygdala in Control of Alcohol-Seeking Behavior 
Marzena Stefaniuk, Anna Beroun, Tomasz Lebitko, Olga Markina, Szymon Leski, Ksenia Meyza, Anna Grzywacz, Jerzy Samochowiec, Agnieszka Samochowiec, Kasia Radwanska, Leszek Kaczmarek 
Biological Psychiatry 
Volume 81, Issue 11, Pages (June 2017)
DOI: /j.biopsych
Copyright © 2017 Society of Biological Psychiatry Terms and Conditions

2. Figure 1
Matrix metalloproteinase-9 (MMP-9) knockout (KO) mice have decreased motivation for alcohol. MMP-9 KO mice (n = 11) and their wild-type (WT) littermates (n = 11) were trained in the IntelliCages. (A) Experimental timeline. (B) IntelliCage setup during the tests. During adaptation and long-term ethanol drinking animals had unlimited access to water or alcohol and water. The dashed line during motivation tests indicates increasing number of nose pokes (NPs) that animal needs to perform in combined fixed ratio (FR) and progressive ratio schedule to obtain access to alcohol. The solid line during withdrawal test indicates no access to alcohol. (C) General activity of mice during experiment. There was no effect of mice genotype on alcohol consumption (two-way analysis of variance [ANOVA]: genotype: [F1,18 = 1.18, p > .05], time [F7,126 = 3.720, p < .01]) and number of NPs performed to all corners (all NPs) during the experiment (two-way ANOVA: genotype [F1,17 = 3.78, p > .06], time [F12,204 = 65.43, p < .0001]). (D) Motivation for alcohol (M1 and M2). MMP-9 KO mice have lower motivation for alcohol as indicated by the number of NPs performed to the alcohol corner (reward NPs; two-way ANOVA: genotype [F1,152 = 8.48, p < .01], time [F7,152 = 0.82, p > .05]) as compared with WT mice in two motivation tests. There was no effect of genotype on NPs performed to the water corner (water NPs) during the tests (two-way ANOVA: genotype [F1,20 = 1.41, p = .24], time [F7,140 = 14.23, p < .0001]). (E) Alcohol withdrawal (W1). MMP-9 KO mice performed fewer reward NPs during withdrawal (W1) when compared with WT animals (two-way ANOVA: genotype [F1,19 = 5.51, p < .05], time [F6,114 = 6.039, p < .0001]). There was no difference between the genotypes in NPs performed to the water corner during the test (two-way ANOVA: genotype [F1,19 = 0.47, p = .49], time [F6,114 = 2.211, p < .05]). Day 0 indicates the day before the test. The effect of genotype: *p < .05. CA, cage adaptation; FAA, free alcohol access; M, alcohol motivation test; NPA, nose poke adaptation; R, relapse; W, withdrawal test; 4–12%, increasing concentration of ethanol.
Biological Psychiatry  , DOI: ( /j.biopsych )
Copyright © 2017 Society of Biological Psychiatry Terms and Conditions

3. Figure 2
Alcohol addiction–related behavior and dendritic spine dynamics in the central amygdala of wild-type (WT) and matrix metalloproteinase-9 (MMP-9) knockout (KO) mice after long-term alcohol drinking in the IntelliCage system. (A) Persistence in alcohol seeking. Reward nose pokes (NPs) per day performed in the nonreward period (withdrawal 2) and before the test (7 days of baseline). WT high (WT-H) persisted/motivated animals performed more NPs to reward than did WT-low (WT-L) persisted/motivated WT animals during withdrawal (W) and baseline (B) (two-way analysis of variance [ANOVA]: effect of groupL/H [F1,14 = 8.101, p = .012], effect of testB/W [F1,14 = 7.173, p = .018]). KO-high (KO-H) persisted/motivated animals performed more nose pokes to reward than did KO-low (KO-L) persisted/motivated animals during withdrawal (two-way ANOVA: effect of groupL/H [F1,16 = 8.472, p = .01], effect of testB/W [F1,16 = 3.1, p = .097]). (B) Example dendrites of L and H persisted/motivated animals per genotype. (C) Density of central amygdala dendritic spines. MMP-9 KO-L mice have lower density of long spines when compared with WT-L animals. MMP-9 KO-H animals have higher density of dendritic spines when compared with MMP-9 KO-L mice (two-way ANOVA: effect of groupL/H [F1,14 = 1.118, p = .3083], effect of genotypeWT/KO [F1,14 = 5.034, p = .0415], followed by Tukey’s post hoc). No such change was observed in population of mushroom (two-way ANOVA: effect of groupL/H [F1,14 = , p = .8662], effect of genotypeWT/KO [F1,14 = , p = .6361]) and stubby spines (two-way ANOVA: effect of groupL/H [F1,14 = 4.427, p = .0539], effect of genotypeWT/KO [F1,14 = 1.745, p = .2077]). (D) Area of central amygdala dendritic spines. WT-H animals have bigger long (two-way ANOVA: effect of groupL/H [F1,15 = 13.24, p = .0024], effect of genotypeWT/KO [F1,15 = , p = .5074], followed by Tukey’s post hoc) and mushroom spines (two-way ANOVA: effect of groupL/H [F1,15 = 8.069, p = .0124], effect of genotypeWT/KO [F1,15 < , p = .9985]) when compared with WT-L animals. No change in size was observed in stubby spines for both WT and KO animals (two-way ANOVA: effect of groupL/H [F1,15 = , p = .3702], effect of genotypeWT/KO [F1,15 = , p = .9610]). WT-H n = 4, WT-L n = 5, KO-H n = 4, KO-L n = 6. *p < .05, **p < .01.
Biological Psychiatry  , DOI: ( /j.biopsych )
Copyright © 2017 Society of Biological Psychiatry Terms and Conditions

4. Figure 3
Exogenous expression of matrix metalloproteinase-9 (MMP-9) in the central nucleus of the amygdala (CeA) increases motivation for alcohol in MMP-9 knockout (KO) mice. Wild-type (WT) and MMP-9 KO mice had lentiviral vectors (LVs) (LV-MMP-9 and LV-green fluorescent protein [GFP] or LV-GFP only) injected into the CeA and were trained to drink alcohol in the IntelliCages. (A) Lentiviral infection in the CeA and microphotography of the CeA transfected with LV-GFP. Scale bar = 500 µm. Gel zymography shows enzymatic activity of MMP-9 and MMP-2 in homogenates from the central amygdala of animals lacking functional MMP-9 (MMP-9 KO) after injection of LVs expressing MMP-9 and GFP or GFP only. (B) Experimental timeline. (C) General activity of mice during the experiment. There was no effect of viral transfection on alcohol consumption by WT mice (two-way analysis of variance [ANOVA] for alcohol consumption: virus [F1,14 = 2.27, p > .05]). There was also no effect of viral transfection on alcohol consumption by KO mice (two-way ANOVA for alcohol consumption: virus [F1,7 = 0.02, p > .05]). (D) Motivation for alcohol (M1 and M2). MMP-9 KO animals with exogenous local expression of MMP-9 (KO-LV-MMP-9, n = 5) in CeA had higher motivation for alcohol than did control MMP-9 KO mice (KO-LV-GFP, n = 4), as indicated by the number of reward nose pokes (NPs) performed during the tests (two-way ANOVA for reward NPs: virus [F1,50 = 28.12, p < .01]). No effect of virus was observed for WT mice on the number of reward NPs (two-way ANOVA for reward NPs: virus [F1,15 = 0.01, p > .05]). WT-LV-GFP n = 8, WT-LV-MMP-9 n = 9. (E) Alcohol withdrawal (W1). KO-LV-MMP-9 mice performed more reward NPs during the nonreward period (W1) when compared with KO-LV-GFP animals (two-way ANOVA: virus [F1,49 = 4.85, p < .05]). There was no difference between WT-LV-GFP and WT-LV-MMP-9 animals in the number of reward NPs during the test (two-way ANOVA for reward NPs: virus [F1,77 = 0.14, p > .05]). Day 0 indicates the day before the tests. For all data mean ± SEM is shown. The effect of the virus: **p < .01. CA, cage adaptation; FAA, free alcohol access; M, alcohol motivation test; NPA, nose poke adaptation; R, relapse; W, withdrawal test; 4–12%, increasing concentration of ethanol.
Biological Psychiatry  , DOI: ( /j.biopsych )
Copyright © 2017 Society of Biological Psychiatry Terms and Conditions

5. Figure 4
Alcohol withdrawal induces glutamatergic silent synapses in the central nucleus of the amygdala (CeA) of wild-type (WT) mice but not in matrix metalloproteinase-9 (MMP-9) knockout (KO) mice. (A) Experimental timeline. (B) Example traces for minimal stimulation protocol in WT mice, showing 5 consecutive sweeps of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor–mediated currents recorded at –60 mV and +45 mV, respectively. The stimulation strength was reduced to obtain both responses and failures (WT water group, bright gray traces: three responses and two failures for both AMPA and NMDA currents). After prolonged withdrawal, the number of AMPA-silent synapses accumulated, which is represented by no failures for NMDA and four failures for AMPA currents in five consecutive trials (WT 7-day withdrawal [WD7] group, dark gray traces). Scale bars = 20 pA, 20 ms. The bar graph shows quantified number of silent synapses. In WT mice but not MMP-9 KO mice, alcohol consumption and extended withdrawal elevate silent synapse number in the CeA (two-way analysis of variance [ANOVA]: genotype [F1,32 = 6.65, p = .0147], alcohol exposure [F3,32 = 6.47, p = .0015]; WT water group n = 4, 1-day withdrawal [WD1] n = 6, WD7 n = 7). Two-hour presentation of alcohol cue decreases the number of silent synapses in CeA (Tukey’s post hoc test of WT WD7 vs. WT WD7+2h cue: p = .013, WD7+2h cue n = 5). MMP-9 KO mice have an elevated baseline level of silent synapses (Mann-Whitney test WT water vs. KO water p = .016), with no effect of alcohol consumption and withdrawal on this parameter (KO water group n = 5, WD1 n = 4, WD7 n = 5, WD7+2h cue n = 4). (C) Averaged example traces of AMPA receptor– and NMDAreceptor–mediated excitatory postsynaptic currents (EPSCs) (recorded at –60 and +45mV, respectively), normalized to the amplitude of AMPA receptor traces, showing a relative increase in NMDA currents in the CeA after a prolonged alcohol withdrawal in WT mice. Scale bars = 50 pA, 25 ms. A bar graph shows the summary of AMPA/NMDA EPSC ratios over all experimental groups. Similar to silent synapse number, in only WT but not in MMP-9 KO animals was alcohol able to alter synaptic efficacy (two-way ANOVA: genotype [F1,33 = 4.645, p = .038]). Extended withdrawal from alcohol caused a decrease of AMPA/NMDA ratio, while exposure to alcohol-related cue elevated this parameter (two-way ANOVA: alcohol exposure [F3,33 = 1.495, p = .234]; Tukey’s post hoc test of WT water vs. WT WD7: p = .034; WD7 vs. WT WD7+2h cue: p = .048, WT water group n = 4, WD1 n = 6, WD7 n = 7, WD7+2h cue n = 5; KO water group n = 5, WD1 n = 4, WD7 n = 5, WD7+2h cue n = 4). *p < .05, **p < .01, ***p < FAA, free alcohol access.
Biological Psychiatry  , DOI: ( /j.biopsych )
Copyright © 2017 Society of Biological Psychiatry Terms and Conditions