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. 2023 Dec 18;26(12):828-839.
doi: 10.1093/ijnp/pyad060.

The Impact of Heroin Self-Administration and Environmental Enrichment on Ventral Tegmental CRF1 Receptor Expression

Ewa Galaj  1 , Eddy D Barrera  2 , Kirk Persaud  3 , Rudolf Nisanov  2 , Apoorva Vashisht  2 , Hindy Goldberg  3 , Nima Patel  3 , Hayley Lenhard  1 , Zhi-Bing You  4 , Eliot L Gardner  4 , Robert Ranaldi  2   3
Affiliations

The Impact of Heroin Self-Administration and Environmental Enrichment on Ventral Tegmental CRF1 Receptor Expression

Ewa Galaj et al. Int J Neuropsychopharmacol. .

Abstract

Background: There is a strong link between chronic stress and vulnerability to drug abuse and addiction. Corticotropin releasing factor (CRF) is central to the stress response that contributes to continuation and relapse to heroin abuse. Chronic heroin exposure can exacerbate CRF production, leading to dysregulation of the midbrain CRF-dopamine-glutamate interaction.

Methods: Here we investigated the role of midbrain CRF1 receptors in heroin self-administration and assessed neuroplasticity in CRF1 receptor expression in key opioid addiction brain regions.

Results: Infusions of antalarmin (a CRF1 receptor antagonist) into the ventral tegmental area (VTA) dose dependently reduced heroin self-administration in rats but had no impact on food reinforcement or locomotor activity in rats. Using RNAscope in situ hybridization, we found that heroin, but not saline, self-administration upregulated CRF1 receptor mRNA in the VTA, particularly on dopamine neurons. AMPA GluR1 and dopamine reuptake transporter mRNA in VTA neurons were not affected by heroin. The western-blot assay showed that CRF1 receptors were upregulated in the VTA and nucleus accumbens. No significant changes in CRF1 protein expression were detected in the prefrontal cortex, insula, dorsal hippocampus, and substantia nigra. In addition, we found that 15 days of environmental enrichment implemented after heroin self-administration does not reverse upregulation of VTA CRF1 receptor mRNA but it downregulates dopamine transporter mRNA.

Conclusions: Overall, these data suggest that heroin self-administration requires stimulation of VTA CRF1 receptors and upregulates their expression in brain regions involved in reinforcement. Such long-lasting neuroadaptations may contribute to continuation of drug use and relapse due to stress exposure and are not easily reversed by EE exposure.

Keywords: CRF; CRF1; RNAscope; addiction; environmental enrichment; heroin; nucleus accumbens; self-administration; ventral tegmental area; western blot.

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Figures

Figure 1.
Figure 1.
A–H show the data from Exp. 1 investigating the role of ventral tegmnetal area (VTA) corticotropin-releasing factor 1 (CRF1) receptors in heroin self-administration (IVSA). (A) the timeline for behavioral experimentation involving intra-VTA infusions of antalarmin (a CRF1 receptor antagonist) during heroin IVSA. (B) Figure with average means of heroin infusions earned during 3 hour- IVSA sessions in the absence or presence of intra-VTA antalarmin. Pharmacological blockade of VTA CRF1 receptors resulted in a significant reduction in heroin intake; *P < .05. (C) A representative event record of a rat self-administering heroin during a baseline (top panel) and test session with antalarmin vehicle. The pattern of heroin IVSA remained similar across the sessions. (D) A representative event record of a rat self-administering heroin during a baseline and test session with 4 µg/0.5 µL/side of antalarmin, showing heroin intake was reduced during the test with antalarmin. (E) Figure with average numbers of active lever presses for food during 1-hour food self-administration sessions in the presence of intra-VTA antalarmin, indicating no significant changes in food-reinforced lever pressing. (F) The average numbers of inactive lever presses during food self-administration, indicating no effect of antalarmin on nonreward-related behavior. (G) Figure with the average total distance travelled during 2 hours in the presence of intra-VTA antalarmin, indicating that antalarmin had no impact on nonspecific behavior. (H) representative image from cannula placement verification conducted after behavioral assessment.
Figure 2.
Figure 2.
A–K show the data from Exp. 2 investigating neuroadaptations in the VTA after chronic heroin or saline IVSA. (A) The timeline of behavioral assessment and RNAscope ISH assay. (B) Average means of heroin or saline IVSA across 15 sessions, indicating an escalating increase of heroin intake across sessions and significant group differences within each session; *P < .05. (C) Representative images taken at 40× showing colocalization of CRF1, dopamine transporter ( DAT), and AMPA glutametergic receptors 1 (GluR1) mRNA in the VTA. A higher expression of VTA CRF1 mRNA was observed in heroin self-administering rats compared with saline rats. (D) In contrast to saline rats, heroin rats had higher numbers of CRF1 puncta per VTA cells; *P < .05 (E) per DAT cells (*P < .05) and (F) per AMPA GluR1-expressing neurons in the VTA (*P < .05). (G) The AMPA GluR1 mRNA expression in the VTA did not change after heroin IVSA. (H) Similar levels of GluR1 were observed in DAT cells of heroin and saline rats. (J) VTA CRF1 expression significantly correlated with the total number of heroin intake (P < .05). (K) There was no correlation between VTA GluR1 mRNA expression and total heroin intake across 15 IVSA sessions.
Figure 3.
Figure 3.
A–F show the data from Exp. 3 investigating corticotropin-releasing factor 1 (CRF1) receptor neuroadaptation after heroin or saline intravenous self-administration (IVSA). Top panels show blot image data from western-blot assay detecting CRF1 (48 kDa) and beta-actin protein (45 kDa). Middle panels show data quantification showing the relative ratio between CRF1 and beta-actin protein expression. Significant upregulation of CRF1 protein was observed in (A) the ventral tegmental area, VTA (*P < .05) and (B) nucleus accumbens, NAc (*P < .05) but not (C) substantia nigra, SN, (D) prefrontal cortex, PFC, (E) dorsal hippocampus, dHippo, or (F) insula. Bottom panels show correlations between relative ratio and heroin intake. A significant correlation was observed between heroin intake and relative ratio of CRF1: beta-actin expression in the NAc (B) but not for other brain regions.
Figure 4.
Figure 4.
A–I show the data from Exp. 4 investigating the impact of environmental enrichment (EE) on corticotropin-releasing factor (CRF1), AMPA glutamatergic receptor 1 (GluR1) and dopamine transporter (DAT) mRNA in the ventral tegmental area (VTA). (A) A timeline of behavioral assessment, EE, and RNAscope assay. (B) Average means of heroin infusions across 15 sessions, indicating a similar pattern of heroin intravenous self-administration (IVSA) between EE and non-EE groups. (C) Representative images taken at 40× showing CRF1, GluR1, and DAT mRNA expression in EE and non-EE rats. (D) The similar levels of CRF1 mRNA on VTA cells, (E) DAT cells, and (F) GluR1-expressing cells were detected in EE and non-EE rats. (G) There was no significant difference between EE and non-EE groups in the GluR1 mRNA puncta expression per VTA cells and (H) per DA cells. (I) EE exposure downregulated DAT mRNA expression in the VTA (*P < .05).
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