Our study revealed a crucial role of mesolimbic DA transmission in mediating the reinforcing properties of opioids. Specifically, we found that the administration of heroin to drug-naïve animals increased both the activity of dopaminergic neurons in the VTA, as well as DA levels in the NAc shell.
We postulate that the heroin-induced changes in mesolimbic DA transmission occurred through a disinhibitory mechanism, whereby heroin targeted GABAergic neurons in the VTA that then
increased the activity of DA neurons in the NAc shell. Finally, we demonstrated that this mechanism appeared engaged at the earliest stage of drug exposure (i.e., after the first self-administration session), before mice were dependent.
5.1 Function of subpopulation located in the medioventral VTA
Several studies have revealed a critical role for VTA afferents in mediating the effects of drugs 228–230; however, the afferents responsible for mediating these effects, as well as the specific neuronal population which drive addiction-like behaviors, remain unclear. The VTA is canonically thought of as a heterogeneous structure comprised of different neuronal populations, with dopaminergic neurons (60-65%) contributing the most to this structure, followed by GABAergic (30-35%), glutamatergic (around 2%), and mixed neuron groups 67,69,70,72,73. Since Schultz’s experiments 78,209,231, mesolimbic DA neurons have been described as a homogeneous group required for reward prediction, error, and learning. Studies over the last decade, however, have revealed the VTA could be even more complex
than previously thought, with heterogeneity presenting in a single neuronal population. Recent findings in both rodents 232–234 and non-human primates 235,236 support this view, as midbrain DA neurons seem to differ in their molecular properties, responses to external stimuli and, thus, in their ability to modulate certain behaviors. It has been reported that DA neuron subpopulations—
identifiable by differences in their basal synaptic properties—can be selectively modified by either aversive or rewarding stimuli depending on their specific brain targets 237. In fact, anatomically and functionally distinct subpopulations can even be observed for VTA DA neurons which project to different NAc subdivisions (i.e., the medial shell, lateral shell, and core) (Lammel et al. 2008, 2012;
Beier et al. 2015). Indeed, a very recent study revealed that motivated behaviors can be regulated by these different NAc subnuclei via either direct inhibition or disinhibition of VTA DA subpopulations
240. Our results support the hypothesis that midbrain DA neurons are not homogenous. In fact, one surprising finding of our study was that we observed a clustering of DA neurons in the ventrolateral division of the VTA that had been activated after only 1 day of heroin self-administration. Another interesting aspect of our study is that the lateral and medial NAc seedings in the VTA are well segregated (we observed only 2.83 ± 0.83% of neurons in the VTA projecting to both parts of the NAc), an observation coherent with the literature 240. We observed a non-negligible amount of neurons (23.6 ± 4.1 %) which were not projecting to the NAc (neither the lateral nor medial shell) and several reasons can explain this observation. First, the infection by the CTB was maybe not efficient enough and it is likely that neurons have not been hit. The other obvious reason is that those neurons are not DA neurons projecting to the NAc. Indeed as we discussed it already in this thesis, the VTA has a wide range of targets (see Introduction, Figure 11) and projecting neurons are not all dopaminergic. What could be done to assess this point would be to re-do the experiment, adding a TH and or GAD staining to fully appreciate the nature of these not-projecting neurons. Based on the literature 71 we can be confident that the projecting neurons positive for CTBs staining are dopamine ones. The same scenario applies for the population of neurons we observed that was only cFos positive (25 ± 7.05%). First if the infection in the NAc was not 100% efficient then some of the cFos-only positive neurons we observed are actually false negative. Moreover as we already said, VTA is innervated by many brain structures, some of them as the LTD have been shown to play a role in reinforcing properties of opioids 241. VTA also have other targets than the NAc. Thus because of this heterogeneous nature in neuronal populations, afferents/efferents and neurotransmitter content these data are not surprising. Taken together our data revealed that DA neurons within the ventrolateral VTA that project mostly to the lateral NAc shell play a key role in the mediating the reinforcing properties of heroin.
5.2 Role of GABA neurons in the VTA
It has been shown that most (50-70%) of the afferents to midbrain DA neurons are GABAergic, and that the inhibitory tone provided by these inputs is a major factor contributing to DA neuron activity
242. Importantly, the removal of tonic inhibition from VTA DA neurons is thought to be the most promising mechanism by which drugs of abuse increase DA neuron activity 86. As recent anatomical studies have shown that the NAc is a major source of GABAergic input to the VTA 239, we considered this region as a prime candidate for mediating the reinforcing properties of heroin.
An alternative structure that could be involved in mediating the reinforcing effects of heroin is the pedunculopontine tegmental nucleus (TPP), as it has been implicated in regulating reward-driven behaviors 238,243,244, and some in vitro data has suggested a functional connection between the VTA and the TPP 245,246. The TPP is a heterogeneous nucleus containing distinct populations of GABAergic, glutamatergic, and cholinergic neurons that project to the midbrain DA system 247,248; however, the majority of TPP projections target the substantia nigra 248,249, while only a small fraction appear go to the VTA 248,249. The discrepancy between the anatomical and behavioral data could be explained by the activation of fibers from nearby regions, such as the LDT, which has been shown to strongly innervate the VTA 241. In agreement with this, it has been reported that activation of the LDT-VTA pathway leads to conditioned placed preference (CPP) and reinforces operant responses 238,244. There is also evidence for a role of the LDT in drug-induced behaviors 28,250.
As the VTA-NAc pathway (versus the TPP-VTA pathway) seemed more likely to underlie the initial reinforcing effects of heroin, we focused on this projection for our study. Thus, after confirming an increase in NAc DA levels following heroin exposure, we evaluated the activity of VTA DA neurons following heroin injection. Using fiber photometry to selectively record DA neurons in the VTA, we observed an increase in activity following the very first heroin exposure.
We also utilized a behavioral paradigm and assessed DA transmission in order to test (in a more specific and refined way) the DA hypothesis that has been challenged for decades. Specifically, we used DREADD technology to inhibit VTA DA neurons and then investigated the consequence of this manipulation on heroin SA behavior. In order to take into consideration the deprived/non-deprived model, we designed two experiments. In the first study, we inhibited DA transmission when mice were well-trained, having been exposed to heroin multiple times. Using this approach, we observed a drastic but reversible (when inhibition was relieved) decrease in the SA behavioral response. In the second study, DA transmission was inhibited in drug-naïve animals (i.e., from the first SA session) and, while this approach prevented the occurrence of SA behavior, we found that the effect was
once again reversible; mice began self-administering heroin as soon as the inhibition of DA transmission was relieved. We then took advantage of a previously designed, drug-independent model of addiction to further assess the necessity of functional DA transmission on the initial reinforcing effects of heroin. Previously, it had been shown that animals readily self-administer heroin during optogenetic activation of VTA DA neurons 87. We hypothesized that if heroin self-administration was dependent on VTA DA neurons activity, an injection of heroin itself would interfere with optogenetic self-stimulation of these neurons. Animals rapidly acquired the optogenetic self-stimulation task and displayed strong behavioral responses to laser stimulation.
After heroin injection, however, their performance decreased in a dose-dependent fashion, with animals receiving the highest dose of heroin showing a complete cessation of optogenetic self-stimulating lever-pressing. In order to ensure that sedation was not the cause of this effect, we tested a separate group of mice for locomotor activity, using the same doses of heroin. As no sedative effects were observed (in contrast, mice displayed increased locomotive activity that even was evident at the highest dose of heroin), our results provide compelling evidence that the reinforcing effects of heroin are dependent on VTA DA neurons activity.
5.3 Critique of the TPP model
A point the present study aimed to highlight is the molecular mechanism underlying the reinforcing properties of opioids, as this is a question that is still debated in the field. While some studies
204,205,208 claim the existence of an alternative model, where the initial phase of heroin exposure falls under the control of the TPP and is thus DA-independent, our data point toward another mechanism.
First, the alternative model does not explain the acute, opioid-induced increase in extracellular NAc DA that we and others 102,154,155 have observed. Indeed, a striking feature of our study was the strong activation of VTA DA neurons—and the immediate subsequent increase of NAc DA—following the very first exposure to heroin. Moreover, the alternatively proposed model does not take into account changes in synaptic strength that have been reported in NAc MSNs (e.g. changes in AMPAR signaling, AMPAR subunit composition, and neurotransmitter release) after only 5 days of i.p. morphine injections 167. Together, these results suggest an acute effect of opioids in the NAc that involves DA at the very earliest stage of opioid exposure. However, acute opioid exposure also exerts effects on VTA DA neurons that should be mentioned. For example, acute opioid injection causes an immediate inhibition of the GABAergic VTA neurons that contain MORs and which make strong synaptic
contacts on the soma and dendrites of DA neurons 103,156,173. Additionally, presynaptic glutamate terminals are inhibited via MOR-mediated arachidonic acid-dependent activation of voltage-sensitive K+ channels 174, and an increase in AMPAR-mediated transmission in DA neurons appears 24 h later
175–177. This increase in AMPAR-mediated transmission is thought to be caused by greater surface expression of AMPARs. In line with these findings, an increase in GluR1 177 can be seen as early as 1 h following a morphine injection 178. Although the mechanisms underlying the insertion of GluR1 subunits and the resulting LTP have not been fully elucidated, they are thought to involve the activation of dopamine D5 receptors 177,179. Taken together, it seems difficult to envisage a model where DA transmission would not be involved in the reinforcing properties of opioids; however, the mechanics of this model remain unknown. The most commonly described actions of opioid are inhibitory. Indeed, stimulation of MORs results in different cellular mechanisms, including the inhibition of adenylyl cyclase (Chieng and Williams 1998; Ingram et al. 1998; Shoji, Delfs, and Williams 1999), activation of potassium conductances—the most commonly observed being the activation of somatodendritic G-protein coupled receptor activated inwardly rectifying K+ (GIRK) channels, which leads to the hyperpolarization of GABAergic interneurons (GIRKs, (Johnson and North 1992; Ingram et al. 1998; van Zessen et al. 2012) or the direct inhibition of GABA release 156,213. Together, this strong body of evidence led us to propose a model of VTA DA neuron activation through the removal of tonic GABAergic inhibition. Indeed, such disinhibitory models have already been proven correct in the hippocampus and other brain areas 254. While most of our experiments selectively targeted DA neurons in the VTA, we also evaluated the effects of heroin on VTA
GABAergic neurons. Specifically, we hypothesized that the inhibition of these neurons would be reinforcing, and that heroin would occlude these reinforcing effects. As described, we observed strong self-inhibition behavior that was easily acquired from the very first session. However, after heroin injection, the performance significantly decreased in a dose-dependent fashion, with the self-inhibition response completely abolished at the highest dose of heroin. Our data demonstrate that the reinforcing effects achieved by ontogenetically self-stimulating GABAergic VTA neurons and by those observed following heroin administration are mediated by shared neural circuits, confirming a disinhibitory mechanism whereby heroin targets GABAergic neurons.
5.4 Critique of pharmacological challenges
In the late 1980s and 1990s, two studies which challenged the DA hypothesis of reward are still valid today. Van Ree and Ramsey 183 attempted to directly test the dopamine hypothesis of opioid reward by blocking DA receptor systems with the D2R antagonist, haloperidol. When administered either systemically or locally into different brain areas in rats, the haloperidol treatments did not block the acquisition of opioid SA behavior. The authors thus concluded that DA is not critically involved in opioid reward. A few years later 188, the same group used identical experimental procedures to examine the involvement of D1 receptors in the reinforcing properties of opioids. In order to do so, they administered the D1 antagonist, SCH23390, either systemically or locally in the NAc (without distinguishing between the subnuclei which constitute the NAc) and looked at the initiation of heroin SA. It appeared that systemic antagonist treatment significantly decreased heroin consumption during the initiation of heroin SA, whereas intra-NAc infusion of the D1 antagonist did not affect heroin intake at any dose. One explanation for discrepancy between systemic and intra-cranial results 189 could be due to a difference in receptor occupancy that is dependent on the way the antagonist is administered. Indeed, it has been reported that, when injected systematically, SCH23390 occupies 72-100% of D1Rs but only 40-60% when administrated intra-cranially. Notably, most of the studies referenced above employ pharmacology and/or lesions and rely solely on equivocal behavioral paradigms (e.g., the CPP) to assess the effects of their manipulations. Their results, therefore, reflect a global net effect which do not take into account potential selective or differential opioid activation of neuronal subpopulations in the NAc or VTA. Recent studies have actually shown that the mesolimbic DA system is more complex than previously thought, and that the system is comprised of distinct anatomical circuits that are differentially regulated depending on the stimuli 72,238,255. In contrast, other studies argue the opposite, postulating the requirement for functional DA transmission in mediating the reinforcing properties of opioids. Mice learn to self-administer MOR agonists directly into the NAc 190,191, and lesioning or inactivating the NAc has been shown to reduce opioid self-administration 192–194. Further, and in favor of a role for dopamine in the reinforcing properties of opioids, electrolytic lesions of the NAc result in significant decreases of intravenous cocaine and morphine self-administration under a progressive ratio schedule of reinforcement (a paradigm used in order to assess drug wanting while avoiding difficulties in
interpretation that can occur with a classic SA paradigm) (Suto, Wise, and Vezina 2011). Adding more complexity to the picture, a recent study using an siRNA strategy reported that impairments of D1aR in the NAc shell prevent the acquisition of cocaine, but not heroin, self-administration 196. However, this study focused on cocaine SA and the distinct involvement of the NAc shell and core divisions in mediating the reinforcing properties of cocaine. Thus, a mechanism to explain the results obtained
with heroin was not supplied. It has also been shown that in the NAc both D1 and D2Rs were activated following heroin SA 198. These results are for sure intriguing and ask for more investigation to fully capture their signification.
5.5 Critique of genetic challenges
At the end of the 1990s, a study reported that mice deficient in the DA transporter (DAT KO mice) were still able to self-administer cocaine 199. This observation presented a huge challenge to the DA hypothesis, and it took a decade for the field to fully understand it. Interestingly, that same year, a study showed 200 that both DAT KO mice and mice deficient in the serotoninergic transporter (SERT) develop cocaine CPP. This observation led to the hypothesis that either DAT or SERT can mediate cocaine reward in the other's absence. In fact, in DAT KO mice (and reciprocally in SERT KO mice), a compensatory reuptake of DA was shown to occur through other monoamine transporters (i.e., SERT and NERT). Moreover, because cocaine blocks the uptake of DA by these transporters, DA was shown to increase on drug exposure, even in the absence of DAT 201. Closure came when an experiment using a knock-in mouse line, which carried a functional DAT that was insensitive to cocaine, showed that the self-administration of cocaine, but not amphetamine, is abolished in these animals 202. The observation that morphine still induced CPP in DA-deficient mice has also challenged the DA hypothesis 203. However, caution should be employed in interpreting the data obtained using DA-deficient animals, as they suffer from severely reduced locomotion and other developmental adaptations, which preclude the testing for late-stage drug-adaptive behaviors.
5.6 Implications for opioid addiction and its treatment
Determining the validity of the DA hypothesis for opioids has some importance with regard to the attempts that have been made to propose a circuit model for addiction, which consider all classes of drugs leading to the same disease. The heterogeneity of the VTA and the ubiquitous presence of MOR in neuronal populations which differ in their neurotransmitter content, as well as the variety of projection targets, makes a unitary model of MOR-driven reward quite difficult to envisage.
However, what seems clear from this study is the necessity and sufficiency of VTA DA neuron
disinhibition in mediating the reinforcing properties of opioids. Untangling the circuits which underlie the reinforcing properties of any drug of abuse will be crucial to propose refined treatments and/or interventions, as well as to design drugs which have the strong antalgic effects of opioids (most potent analgesics in clinical uses) without the rewarding, euphoric affects that lead to misuse and abuse. This constitutes a true scientific challenges as both rewarding and analgesic actions of opioids depend upon actions at MORs.