Extracellular matrix abnormalities in the hippocampus of subjects with substance use disorder

Contextual triggers are significant factors contributing to relapse in substance use disorders (SUD). Emerging evidence points to a critical role of extracellular matrix (ECM) molecules as mediators of reward memories. Chondroitin sulfate proteoglycans (CSPGs) are a subset of ECM molecules that form perineuronal nets (PNN) around inhibitory neurons. PNNs restrict synaptic connections and help maintain synapses. Rodent models suggest that modulation of PNNs may strengthen contextual reward memories in SUD. However, there is currently a lack of information regarding PNNs in the hippocampus of people with SUD as well as how comorbidity with major depressive disorder (MDD) may affect PNNs. We used postmortem hippocampal tissues from cohorts of human and nonhuman primates with or without chronic alcohol use to test the hypothesis that PNNs are increased in subjects with SUD. We used histochemical labeling and quantitative microscopy to examine PNNs, and qRT-PCR to examine gene expression for ECM molecules, synaptic markers and related markers. We identified increased densities of PNNs and CSPG-labeled glial cells in SUD, coinciding with decreased expression of the ECM protease matrix metalloproteinase 9 (Mmp9), and increased expression for the excitatory synaptic marker vesicle associated membrane protein 2 (Vamp2). Similar increases in PNNs were observed in monkeys with chronic alcohol self-administration. Subjects with MDD displayed changes opposite to SUD, and subjects with SUD and comorbid MDD had minimal changes in any of the outcome measures examined. Our findings demonstrate that PNNs are increased in SUD, possibly contributing to stabilizing contextual reward memories as suggested by preclinical studies. Our results also point to a previously unsuspected role for CSPG expression in glial cells in SUD. Evidence for increased hippocampal PNNs in SUD suggests that targeting PNNs to weaken contextual reward memories is a promising therapeutic approach for SUD, however comorbidity with MDD is a significant consideration.


Quantitative polymerase chain reaction
RNA was extracted from 14 µm hippocampal cryosections using the RNeasy Minikit (cat# 74104, Qiagen, NL) according to the manufacturer's instructions.On-column DNAse digestion was carried out using the RNAse-free DNAse set (#79254, Qiagen, NL), per manufacturer's guide.Complementary DNA (cDNA) was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA).For each reaction, 0.5 μl of cDNA (1:3 diluted) was placed in a 10 μL reaction containing 5 μL of SYBR Green PCR Master Mix (Applied Biosystems) and 10 pmol of each primer (Invitrogen, United States) or FastStart TaqMan Probe Master and 1x Taqman primers (ThermoFisher).The primers used are listed in Supplemental Table 2. Custom-made assays were tested for specificity and resulted in a single band of expected size.All reactions were performed in triplicate using 384-well optical reaction plates (Life Technologies, United States) on an Applied Biosystems detection system (QuantStudio 5, Applied Biosystems, Life Technologies, United States).Reactions were performed with an initial ramp time of 10 min at 95°C, and 40 subsequent cycles of 15 s at 95°C and 1 min at 60°C.For negative controls for the qPCR reactions, non-template control (cDNA was omitted) and no-RT control (reverse transcriptase excluded from cDNA synthesis reaction) were run on each plate.Relative concentrations of the transcripts of interest were calculated with comparison to a standard curve made with dilutions of cDNA from a pooled sampling of all the subjects.
Values for the transcripts of interest were normalized to the geometric mean of B2M, ACTB, GAPDH and PPIA values for the same samples.Data were collected by QuantStudio Design and Analysis software v1.5.1.

Figure S1 .
Figure S1.Major depressive disorder duration and severity impacts ECM parameters in the human hippocampus.(A-E) PNNs in CA1 (A-B), bulk hippocampal PVB (C), and PNNs and WFA glia in the DG GCL plotted by the duration of MDD in subjects with MDD (blue) and MDD/SUD (purple).A significant positive correlation was observed in subjects with MDD/SUD in respect to CA1 PNNs in S.O. and S.P., while a negative

Figure S2 .
Figure S2.Effects of antidepressants on ECM parameters.No significant effects of antidepressants in the toxicology report were detected for CA1 SO PNNs (A), CA4 WFA+glia (B), or MMP9 mRNA (C).A significant increase in GFAP gene expression was observed in subjects with SUD/MDD with antidepressants in the toxicology report compared to SUD/MDD subjects without (D).Graphs represent mean of each group, black circles represent values for individual subjects, and black lines represent 95% confidence intervals.No significant correlations were detected in subjects with SUD/MDD and subjects with MDD for antidepressant equivalent amount per last month of life in grams with CA1 SO PNN densities, CA4 WFA-glia densities, MMP9 mRNA, or GFAP mRNA (E-H).

Figure S3 .
Figure S3.Effect of AUD duration on CA1 SO PNNs and Chsy1 gene expression.(A) No correlation was observed between CA1 SO PNNs and AUD duration in subjects with either SUD or SUD/MDD.(B) A negative correlation was observed between Chsy1 gene expression and AUD duration in subjects with SUD/MDD.No correlation was observed in subjects with SUD.

Figure S4 .
Figure S4.Effects of drugs in toxicology reports on ECM-related transcripts, PNNs and WFA + glia.(A,B, and C) In subjects with SUD, cocaine in the toxicology report was associated with a decrease in Pvb and an increase in Ctss gene expression.No significant changes were observed for Acan mRNA.(D) Subjects with SUD and SUD/MDD who had a cocaine history had a lower density of PNNs compared to SUD and SUD/MDD subjects who did not have a cocaine use history.(E-H) Effects of alcohol in the toxicology report in control, SUD, SUD/MDD, and MDD subjects.No statistically significant effects were observed in any diagnostic group (Control and MDD groups did not have adequate numbers of subjects with ethanol in the toxicology report to perform statistical comparisons).

Figure S5 .
Figure S5.Multiplex immunofluorescence detects WFA labeling in GFAP+ astrocytes.(A-I) High-magnification images of WFA (A, D, and G), GFAP (B, E, H) and merged WFA/GFAP (C, F, I) demonstrates co-localization of WFA within astrocytes and their processes (yellow), scalebars = 50 m.(J-L) Low magnification images depicting some WFA glial cells with lack of GFAP overlap, possibly reflecting fine processes of astrocytes with low GFAP levels, scalebar = 100 m.

Table S3 . Hippocampal Gene Expression Measurements Across Diagnostic Groups.
Values represent normalized gene expression.

S8: Basic demographic information for all diagnosis groupsTable S5 :
Basic demographic information for subjects with substance use disorder

Table S6 :
Basic demographic information for subjects with major depressive disorder

Table S7 :
Basic demographic information for subjects with comorbid substance use disorder and major depressive disorder

Table S8 :
Basic demographic information for unaffected control subjects

S11: Substance use information for all diagnostic groupsTable S9 :
Substance use information for subjects with substance use disorder

Table S11 :
Substance use information for subjects with comorbid substance use disorder and major depressive disorder

Table S12 :
Substance use information for unaffected control subjects