Neuroplasticity transcript profile of the ventral striatum in the extinction of opioid-induced conditioned place preference

Abstract

Persistent drug-seeking behavior has been associated with deficits in neural circuits that regulate the extinction of addictive behaviors. Although there is extensive data that associates addiction phases with neuroplasticity changes in the reward circuit, little is known about the underlying mechanisms of extinction learning of opioid associated cues. Here, we combined morphine-conditioned place preference (CPP) with real-time polymerase chain reaction (RT-PCR) to identify the effects of extinction training on the expression of genes (mRNAs) associated with synaptic plasticity and opioid receptors in the ventral striatum/nucleus accumbens (VS/NAc). Following morphine extinction training, we identified two animal subgroups showing either extinction (low CPP) or extinction-resistance (high CPP). A third group were conditioned to morphine but did not receive extinction training (sham-extinction; high CPP). RT-PCR results showed that brain derived neurotrophic factor (Bdnf) was upregulated in rats showing successful extinction. Conversely, the lack of extinction training (sham-extinction) upregulated genes associated with kinases (Camk2g), neurotrophins (Ngfr), synaptic connectivity factors (Ephb2), glutamate neurotransmission (Grm8) and opioid receptors (μ1, Δ1). To further identify genes modulated by morphine itself, comparisons with their saline-counterparts were performed. Results revealed that Bdnf was consistently upregulated in the extinction group. Alternatively, widespread gene modulation was observed in the group with lack of extinction training (i.e. Drd2, Cnr1, Creb, μ1, Δ1) and the group showing extinction resistance (i.e. Crem, Rheb, Tnfa). Together, our study builds on the identification of putative genetic markers for the extinction learning of drug-associated cues.

Keywords: Bdnf; Conditioned place preference; Genes; Learning; Morphine; Reward.

Figure 1.. Extinction of morphine conditioned place preference.

(A) Diagram showing the protocol for morphine conditioning and extinction. (B) A schematic drawing showing the saline-treated (left panel) and morphine-treated (right panel) animal groups throughout the CPP protocol. (C) A pre-conditioning test (baseline) established the non-preferred (drug-paired) side for each animal. After conditioning training, a post-conditioning preference test (conditioning test) was performed. An extinction test was performed to calculate the rate of extinction learning. Two subgroups were observed and separated from the extinction-trained rats, the morphine/extinction and morphine/extinction-resistant groups. **p< 0.01. Saline-extinction: n= 7; Saline/sham-extinction: n= 7; Morphine/extinction: n= 9; Morphine/extinction-resistant: n= 9; Morphine/sham-extinction: n= 9. Data is shown as mean and SEM.

Figure 2.. Extinction-dependent modulation of synaptic plasticity genes.

(A) The morphine/extinction group showed a fold-change increase of Bdnf, as compared to the morphine/sham-extinction group. The morphine/sham-extinction group showed increased expression of Ngfr (B) and Camk2g (C), as compared to both morphine/extinction and morphine/extinction-resistant. Similarly, the lack of extinction training increased the expression of Ephb2 (D) and Grm8 (E), as compared only to morphine/extinction-resistant. *p<0.05; **p<0.01. Morphine/extinction: n= 5; Morphine/extinction-resistant: n= 5; Morphine/sham-extinction: n= 5. Data is shown as mean and SEM.

Figure 3.. Volcano plots for extinction and sham-extinction groups.

(A) Morphine/extinction vs. saline-extinction. (B) Morphine/extinction-resistant vs. saline-extinction. (C) Morphine/sham-extinction vs. saline/sham-extinction. The vertical lines correspond to 1-fold up (to the right) and down (to the left), respectively. Horizontal lines represent a p-value of 0.05. Red dots above and to the right of the horizontal and vertical lines, respectively, represent a significant gene upregulation. Green dots above and to the left of the horizontal and vertical lines, respectively, represent a significant gene downregulation. Saline-extinction: n=5; Saline/sham-extinction: n= 4; Morphine/extinction: n= 5; Morphine/extinction-resistant: n= 5; Morphine/sham-extinction: n= 5.

Figure 4.. Extinction-dependent modulation of gene transcripts for opioid receptors.

Animals in the morphine/sham-extinction group showed a fold-change increase in the expression of transcripts for mu (μ₁ and delta (Δ₁) opioid receptors, as compared to both morphine/extinction and morphine/extinction-resistant. **p< 0.01. Morphine/extinction: n= 5; Morphine/extinction-resistant: n= 5; Morphine/sham-extinction: n= 5. Data is shown as mean and SEM.

Figure 5.. Venn diagram summarizing extinction-dependent genes that were modulated in morphine-treated rats.

The name and number of genes are shown in each extinction learning phenotype. Morphine/extinction (3 genes: Bdnf, Gria 4, Reln); Morphine/extinction-resistant (3 genes: Crem, Rheb, Tnfa); Morphine/sham-extinction (27 genes-see figure). Shared genes between Morphine/extinction and Morphine/sham-extinction groups are also shown (1 gene; Gria4). ↑= upregulation and ↓= downregulation.

Figure 6.. Extinction-dependent network of genes in morphine-treated rats.

(A) Network of genes associated in morphine/extinction (score of 9; network associated to behavior, cell-to-cell signaling and interaction and nervous system development and function), (B) morphine/extinction-resistant (score of 9; network associated to cell-mediated immune response, cellular movement, hematological system development and function (C) morphine/sham-extinction groups (score of 25; network associated to behavior, cell-to-cell signaling and interaction, cell morphology, nervous system development and function). All measures were obtained using Ingenuity Pathway Analysis (IPA). Direct interactions are represented as solid lines, whereas indirect interactions appear as dotted lines. Upregulated and downregulated genes are represented by green and red lines, respectively.