Extracellular matrix plasticity and GABAergic inhibition of prefrontal cortex pyramidal cells facilitates relapse to heroin seeking

Abstract

Successful treatment of drug addiction is hampered by high relapse rates during periods of abstinence. Neuroadaptation in the medial prefrontal cortex (mPFC) is thought to have a crucial role in vulnerability to relapse to drug seeking, but the molecular and cellular mechanisms remain largely unknown. To identify protein changes that contribute to relapse susceptibility, we investigated synaptic membrane fractions from the mPFC of rats that underwent 21 days of forced abstinence following heroin self-administration. Quantitative proteomics revealed that long-term abstinence from heroin self-administration was associated with reduced levels of extracellular matrix (ECM) proteins. After extinction of heroin self-administration, downregulation of ECM proteins was also present in the mPFC, as well as nucleus accumbens (NAc), and these adaptations were partially restored following cue-induced reinstatement of heroin seeking. In the mPFC, these ECM proteins are condensed in the perineuronal nets that exclusively surround GABAergic interneurons, indicating that ECM adaptation might alter the activity of GABAergic interneurons. In support of this, we observed an increase in the inhibitory GABAergic synaptic inputs received by the mPFC pyramidal cells after the re-exposure to heroin-conditioned cues. Recovering levels of ECM constituents by metalloproteinase inhibitor treatment (FN-439; i.c.v.) prior to a reinstatement test attenuated subsequent heroin seeking, suggesting that the reduced synaptic ECM levels during heroin abstinence enhanced sensitivity to respond to heroin-conditioned cues. We provide evidence for a novel neuroadaptive mechanism, in which heroin self-administration-induced adaptation of the ECM increased relapse vulnerability, potentially by augmenting the responsivity of mPFC GABAergic interneurons to heroin-associated stimuli.

Figure 1

Acquisition of heroin self-administration and mPFC dissection. (a) Heroin self-administrating rats developed a stable preference for the active hole, whereas active hole responses remained low for saline self-administration rats (data not shown). Response increased for heroin self-administrating rats when the fixed ratio (FR) was doubled (FR1, FR2, FR4), indicative of a motivational drive to self-administer heroin. Inactive hole responses remained low for both heroin and saline rats during all the sessions. Data represent mean±SEM of responses in active (heroin-paired) or inactive (unpaired) holes. (b) Approximate anatomical dissection limits of the mPFC (between bregma 3.7–2.7) are depicted by the hatched area.

Figure 2

Protein level changes of ECM proteins Bcan and Tnr confirmed by immunoblotting. (a) Synaptic protein (Syn) and total protein (Total) fractions were isolated from the mPFC of a group of animals (independent of iTRAQ experiment) and analyzed by immunoblotting using antibodies against Tnr and Bcan. Representative examples are shown. Levels of immunodetected bands were normalized to proteins on a coomassie (coom)-stained gel that was run in parallel. (b) Optical densities (blot, coomassie) were analyzed and set to the saline control. A significant decrease was detected in the level of Tnr 160 kDa isoform and Bcan 145 kDa isoform in synaptic fractions of the mPFC. Tnr and Bcan protein levels were unaltered in the total mPFC homogenate. (c) The mRNA level of Tnr and Bcan was unaltered in the mPFC during forced abstinence from heroin self-administration. All data are mean±SEM. ^*p<0.05 (Student t-test).

Figure 3

Cue-induced reinstatement of heroin seeking. Exposure to the compound visual and auditory cue reinstated responding in the active (previously heroin-paired) hole. Bars represent mean number of nose pokes±SEM. ^*p<0.01 (Students t-test) for between group difference of responses in active hole and within group difference of active and inactive responses.

Figure 4

ECM proteins are also downregulated after extinction of heroin self-administration. We compared levels of Bcan and Tnr proteins in synaptic fractions of the mPFC, NAc, and dorsal striatum (dStriatum) between animals that underwent saline or heroin extinction, and heroin extinction animals that were re-exposed to heroin-conditioned cues (30 min). Representative examples are depicted on the left side. (a) We observed a significant reduction in the synaptic protein levels of Bcan (145 kDa; p<0.05) and a similar (non-significant) trend for decrease in Tnr 180 and 160 kDa (p>0.05) levels after extinction of heroin self-administration. The abundance of Bcan 145 kDa did not differ between saline animals and heroin animals that were cue-exposed. Similar to levels during abstinence from heroin self-administration, the level of the Bcan 120 kDa isoform was not altered. (b) In the NAc, the levels of Tnr 180 and 160 kDa were significantly (p<0.001) different, with post-hoc analysis revealing a significant difference between all groups. The level of Bcan 145 kDa followed a similar pattern but ANOVA analysis only reached near significance (p=0.074). (c) Abundance of all Bcan and Tnr isoforms in synaptic fractions of the dorsal striatum was not significantly different. All data are mean±SEM. ^*p<0.05 (one-way ANOVA).

Figure 5

Immunohistochemical identification of ECM proteins in the mPFC. (a) Colocalization of Tnr and Bcan in perineuronal nets in the mPFC. Sections were double-stained using Tnr (green) and Bcan (red) specific antibodies and analyzed by confocal microscopy. Immunoreactivity of Tnr and Bcan is colocalized (merged image) in perineuronal nets (white arrows) surrounding a subset of neurons in the anterior cingulate, prelimbic, and infralimbic area of the mPFC. (b+c) Biotinylated-WFA (green) was used to detect chondroitin sulfate proteoglycan-containing perineuronal nets in combination with a PV-specific antibody (red) or CAMKIIα antibody (red). Throughout all areas of the mPFC, perineuronal nets were associated with the majority of PV-expressing interneurons (b), but never with CAMKII-positive pyramidal neurons (c). Scale bar: 20 μm.

Figure 6

Cue-induced enhancement of GABAergic inhibition of mPFC pyramidal neurons. (a) Example traces of whole-cell recordings from mPFC pyramidal neurons in control and heroin self-administration animals (n=5 per group) that were re-exposed (cue) or not exposed (no cue) to the cues. Number of neurons in each group: Heroin cue, n=20; Heroin no cue, n=20; Saline cue, n=17; Saline no cue, n=16. (b) Average IPSC frequency in all neurons recorded during a 3-min period (10 s bins). (c) Summary data and statistical analysis: average IPSC frequency was significantly higher in heroin animals on cue re-exposure (^*p=0.007). IPCS amplitude and decay time constant remained unaltered (p>0.05) on cue re-exposure. Data represent mean±SEM.

Figure 7

FN-439 treatment attenuates cue-induced heroin seeking and increased ECM protein levels in the mPFC. (a) ICV injection of FN-439-attenuated cue-induced heroin seeking as compared with vehicle (ACSF; n=7–9 per treatment). Nose-poking in the inactive hole was not altered by FN-439 treatment (data not shown). (b) Three hours after the last reinstatement test, synaptic fractions were isolated from the mPFC of these animals and analyzed with immunoblotting. (c) A significant increase was detected in the levels of Tnr 160 kDa and Bcan 145 kDa isoform in the mPFC. (d) Expression of Tnr and Bcan in the NAc was increased after FN-439 treatment, but did not reach significance (p>0.05). ^*p<0.05. Data represent mean±SEM.