10 June 2024

Opioid aversion switch found in the brains of mice

American, Canadian and French researchers conducted a series of experiments on mice and found a population of atypical glutamatergic neurons in the ventral prefrontal cortex, which are responsible for switching between the reinforcing and aversive effects of opioids. A report of the work is published in the journal Science.

The action of opioids on the brain's reward system is largely due to stimulation of inhibitory mu-opioid receptors in GABAergic nerve endings that inhibit the activity of dopaminergic neurons in the ventral covering area (VTA) of the brain. This results in increased dopaminergic transmission in the contiguous nucleus and other components of the mesolimbic system responsible for feelings of pleasure and reinforcement. Mu-opioid receptors are also present in several other brain structures where dopamine receptors are absent, but they are much less studied. Observations show that the administration of opioids in standard doses can paradoxically induce aversion rather than reinforcement. The mechanisms of such an effect are largely unknown, yet it may play a significant role in the development of opioid abuse and dependence.

To study them, Paul Kenny of the Icahn School of Medicine and colleagues from Canada, the US and France began by injecting mice with the opioid oxycodone or saline solution and mapping the entire brain for the protein c-Fos, an early marker of neuronal excitation. This identified 28 brain regions with a high density of mu-opioid receptors, including the dorsal peduncular nucleus (DPn), a largely unexplored region of the ventral prefrontal cortex that the researchers focused further attention on.

They used FosTRAP2 mice with an optogenetic construct for targeted optical stimulation of only those DPn neurons whose activity increased after oxycodone injection. When they were stimulated, the mice began to avoid taking the drug, meaning that DPn is probably involved in the regulation of the negative response to opioids. Tracing the axons of these neurons throughout the brain using fluorescent tags showed that they innervate neurons of the parabrachial nuclei (PBn), structures that are involved in respiratory depression and other physiological responses to opioid administration, as well as in their avoidance. MAP-Seq mapping of the connectome of individual cells confirmed this and showed that many of them also have projections in the VTA.

Spatial transcriptome analysis of DPn cells and surrounding cortical areas revealed a rare population of pyramidal neurons in this nucleus expressing vesicular glutamate transporter 2 (DPnvGlut2 neurons), characteristic of subcortical glutamatergic neurons. Tracing of their axons showed that these neurons project to PBn, and their optical stimulation induced avoidance behaviour, reversible by oxycodone injection. RNA sequencing of individual cell nuclei and fluorescence in situ hybridisation determined that DPnvGlut2 neurons express mu-opioid receptors (in the cortex these are characteristic of inhibitory GABAergic interneurons rather than excitatory glutamatergic interneurons).

Electrophysiological recording of the activity of DPnvGlut2 neurons showed that opioids reduce their excitability. At the same time, optical stimulation of their endings increased glutamatergic excitatory impulse in PBn, while opioids inhibited it. Genetic knockout of mu-opioid receptors in DPn neurons did not affect the locomotor stimulatory effect of oxycodone (mediated by the mesolimbic system), but caused mice to avoid the drug (from which they received pleasure in the presence of these receptors). Additional experiments showed that stimulation of DPnvGlut2 neurons increased the manifestations of withdrawal in opioid-dependent mice, while switching off these cells attenuated such symptoms.

Thus, the population of glutamatergic neurons DPn, expressing mu-opioid receptors and innervating PBn, serves as a key regulator of reinforcement and avoidance reactions during the reception of opioids, which underlie the propensity to form and consolidate opioid dependence, the authors conclude.

Earlier British researchers analysed the activity of brain regions in volunteers and concluded that the strength of functional connections between the ventromedial part of the caudate nucleus (one of the structures of the brain reward system) and different parts of the prefrontal cortex is responsible for the propensity to dependence on psychoactive substances and resistance to it.

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