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

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.

Environmental enrichment facilitates electric barrier induced heroin abstinence after incubation of craving in male and female rats.

BACKGROUND: Treatment strategies that aim to promote abstinence to heroin use and reduce vulnerability to drug-use resumption are limited in sustainability and long-term efficacy. We have previously shown that environmental enrichment (EE), when implemented after drug self-administration, reduces drug-seeking and promotes abstinence to cocaine and heroin in male rats. Here, we tested the effects of EE on abstinence in an animal conflict model in males and females, and after periods where incubation of craving may occur. METHODS: Male and female rats were trained to self-administer heroin followed by 3 or 21 days of a no-event-interval (NEI). Following NEI, rats were permanently moved to environmental enrichment (EE) or new standard (nEE) housing 3 days prior to resuming self-administration in the presence of an electric barrier adjacent to the drug access lever. Electric barrier current was increased daily until rats ceased self-administration. RESULTS: We found that 21 days of NEI led to significantly greater heroin self-administration and a trend toward shorter latencies to emit the first active lever press in the first abstinence session compared to 3 days of NEI. EE, when compared to nEE, led to longer latencies in the first abstinence session. Also, EE groups of both sexes and in both NEIs achieved abstinence criteria in significantly fewer numbers of sessions. CONCLUSIONS: EE facilitates abstinence in males and females and after periods where incubation of craving may occur. This suggests that EE may benefit individuals attempting to abstain from heroin use and may aid in the development of long term treatment strategies.

Muscarinic and NMDA Receptors in the Substantia Nigra Play a Role in Reward-Related Learning.

BACKGROUND: Reward-related learning, where animals form associations between rewards and stimuli (i.e., conditioned stimuli [CS]) that predict or accompany those rewards, is an essential adaptive function for survival. METHODS: In this study, we investigated the mechanisms underlying the acquisition and performance of conditioned approach learning with a focus on the role of muscarinic acetylcholine (mACh) and NMDA glutamate receptors in the substantia nigra (SN), a brain region implicated in reward and motor processes. RESULTS: Using RNAscope in situ hybridization assays, we found that dopamine neurons of the SN express muscarinic (mACh5), NMDA2a, NMDA2b, and NMDA2d receptor mRNA but not mACh4. NMDA, but not mACh5, receptor mRNA was also found on SN GABA neurons. In a conditioned approach paradigm, rats were exposed to 3 or 7 conditioning sessions during which light/tone (CS) presentations were paired with delivery of food pellets, followed by a test session with CS-only presentations. Intra-SN microinjections of scopolamine (a mACh receptor antagonist) or AP-5 (a NMDA receptor antagonist) were made either prior to each conditioning session (to test their effects on acquisition) or prior to the CS-only test (to test their effects on expression of the learned response). Scopolamine and AP-5 produced dose-dependent significant reductions in the acquisition, but not performance, of conditioned approach. CONCLUSIONS: These results suggest that SN mACh and NMDA receptors are key players in the acquisition, but not the expression, of reward-related learning. Importantly, these findings redefine the role of the SN, which has traditionally been known for its involvement in motor processes, and suggest that the SN possesses attributes consistent with a function as a hub of integration of primary reward and CS signals.

Comparative in situ hybridization protocols in zebrafish.

In situ hybridization is a commonly used technique in molecular biology to assess the temporal and spatial expression of a given gene. As a long and labor-intensive protocol, double in situ hybridization, which detects two genes in series, is challenging and can require a lot of troubleshooting. Optional additives, polyvinyl alcohol and dextran sulfate, were tested in a standard in situ hybridization protocol and several colorimetric stain pairings using double in situ hybridization in zebrafish embryos. Optional additives can improve staining time and reduce nonspecific background. Nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (BCIP) + Fast Red/BCIP was the most effective stain pairing. As a proof-of-concept, this work shows that Cabin1 and atoh1b are expressed in distinct regions of the developing zebrafish brain.