Dual action of ketamine confines addiction liability

This study in mice shows that ketamine does increase dopamine levels in the brain (nucleus accumbens) but doesn’t lead to synaptic plasticity (e.g. as seen with cocaine). Thus, the addiction liability of ketamine is (relatively) limited (not taking into account social factors).

Abstract

“Ketamine is used clinically as an anaesthetic and a fast-acting antidepressant, and recreationally for its dissociative properties, raising concerns of addiction as a possible side effect. Addictive drugs such as cocaine increase the levels of dopamine in the nucleus accumbens. This facilitates synaptic plasticity in the mesolimbic system, which causes behavioural adaptations and eventually drives the transition to compulsion. The addiction liability of ketamine is a matter of much debate, in part because of its complex pharmacology that among several targets includes N-methyl-D-aspartic acid (NMDA) receptor (NMDAR) antagonism. Here we show that ketamine does not induce the synaptic plasticity that is typically observed with addictive drugs in mice, despite eliciting robust dopamine transients in the nucleus accumbens. Ketamine nevertheless supported reinforcement through the disinhibition of dopamine neurons in the ventral tegmental area (VTA). This effect was mediated by NMDAR antagonism in GABA (γ-aminobutyric acid) neurons of the VTA, but was quickly terminated by type-2 dopamine receptors on dopamine neurons. The rapid off-kinetics of the dopamine transients along with the NMDAR antagonism precluded the induction of synaptic plasticity in the VTA and the nucleus accumbens, and did not elicit locomotor sensitization or uncontrolled self-administration. In summary, the dual action of ketamine leads to a unique constellation of dopamine-driven positive reinforcement, but low addiction liability.”

Authors: Linda D. Simmler, Yue Li, Lotfi C. Hadjas, Agnès Hiver, Ruud van Zessen & Christian Lüscher

Summary

Ketamine is a club drug that is classified as a schedule III substance by the US Food and Drug Administration and as a class B drug in the UK, owing to its putative addiction liability. However, long-term effects of ketamine on mesolimbic reward circuits and behaviour remain elusive. Cocaine potentiates glutamatergic synapses onto dopamine neurons in the ventral tegmental area (VTA), which leads to stronger excitatory synapses onto type-1 dopamine receptor (D1R)-expressing medium spiny neurons in the NAc and eventually synaptic plasticity in the dorsal striatum.

Reinforcement and dopamine increase in the NAc

Ketamine enhanced locomotion similarly to cocaine, and produced dopamine transients in the NAc that were similar in magnitude, but shorter in duration. Ketamine was also rewarding and reinforcing, and increased the area under the curve in a dose-dependent manner.

Disinhibition of VTA dopamine neurons

Ketamine increased the activity of dopamine neurons in the VTA for 5 min, whereas cocaine decreased the activity of dopamine neurons in the VTA. Ketamine induced a strong and sustained inhibition of VTA GABA neurons, whereas cocaine did not affect the activity of these neurons.

We first removed the NMDARs from the VTA GABA cells by using a Cre-dependent CRISPR-SaCas9 knock-out strategy. This led to a loss of ketamine-induced activity in the VTA GABA cells.

Ketamine had a small effect on cocaine-elicited dopamine transients in the NAc, possibly because of an enhanced baseline activity of dopamine neurons. Ketamine also partially occluded laser-induced disinhibition of VTA GABA neurons.

We found that ketamine-evoked dopamine transients and dopamine neuron activity decayed within minutes, whereas fentanyl caused a long-lasting, only slowly decaying NAc dopamine transient through disinhibition. We also found that fluphenazine-N-mustard reduced the cocaine-induced auto-inhibition of dopamine neurons.

Fast off-kinetics and NMDAR antagonism

We tested whether ketamine induces synaptic plasticity by looking for Ca2+-permeable AMPARs in VTA dopamine neurons. Ketamine did not induce the inward rectification that is typical of the presence of such non-canonical AMPARs, in contrast to cocaine.

We repeatedly infused ketamine or cocaine into the NAc to increase the levels of dopamine, but this did not increase the rectification index at excitatory synapses onto VTA dopamine neurons. This suggests that ketamine antagonizes NMDARs, which may be involved in the lack of effect of ketamine on synaptic plasticity.

Ketamine, at concentrations that correspond to the expected levels in the brain in mice, strongly inhibited NMDAR-mediated synaptic currents and prevented long-term potentiation in acute brain slices.

No accumbal drug-evoked plasticity

After a single injection of ketamine, there was no increase of FOS-positive D1-MSNs, and no drug-evoked plasticity was observed in the NAc D1-MSNs. However, the AMPA/NMDA ratio decreased at mPFC-to-NAc D1-MSN synapses, in line with previous reports33,34. In an attempt to test for addiction criteria, we allowed mice daily four-hour-long access to ketamine, increasing the lever press ratio (fixed ratio; FR) every four days. We observed that mice reduced self-administration once FR2 was introduced, speaking to the low motivation for ketamine self-administration.

Discussion

Ketamine increases the activity of dopamine neurons in the ventral tegmental area (VTA) and NAc, but does not evoke drug-adaptive synaptic plasticity, long-term locomotor sensitization or uncontrolled self-administration.

Ketamine inhibits VTA GABA interneurons by reducing NMDAR excitation, which in turn causes the inhibition of pyramidal cells. Ketamine also inhibits VTA dopamine neurons by reducing D2R excitation, which in turn causes hyperpolarization of VTA dopamine neurons.

Ketamine evoked no synaptic plasticity in the mesolimbic dopamine system or long-term locomotor sensitization. This suggests that the addiction liability of ketamine is low.

With a single dose of ketamine, the fast off-kinetics of accumbal dopamine were insufficient to cause plasticity in the VTA, and with repeated intravenous infusions that cause prolonged enhanced levels of dopamine, NMDAR blockade prevented plasticity.

Ketamine indirectly acts on the dopamine system through circuit effects from local GABA neurons, and the absence of drug-adaptive synaptic plasticity strongly indicates that ketamine’s addiction liability is limited by its pharmacology.

Online content

C57BL/6J (wild-type) mice were used, as well as Drd1-Tomato, DAT-Cre, GAD-Cre and VGat-Cre mouse lines. All mice were group housed except for those used for self-administration experiments.

Drugs

Ketamine, fentanyl, cocaine and FNM were dissolved in sterile 0.9% NaCl (saline) and used for intravenous administration.

Acute hyperlocomotion and behavioural sensitization

Mice were habituated to the behaviour room, handling and intraperitoneal injections on three days before testing. On testing days, mice underwent room habituation for a minimum of 1 h, were individually transferred into a testing arena and were i.p. injected with saline, ketamine or cocaine.

Drug self-administration

Jugular vein catheters were implanted under anaesthesia and mice were treated with amikacin for 5 days. Catheters were flushed daily with heparin.

Mice were mildly food-deprived for one night before the first self-administration session to increase exploratory behaviour. They were placed individually into operant boxes with an active and an inactive lever and trained on a fixed ratio (FR) 1 schedule for the acquisition sessions.

Hotplate test

The mice were placed on a pre-heated hotplate apparatus 0.5-1 h before and immediately after ketamine infusions. The latency to hotplate was calculated.

Stereotactic surgeries

Standard stereotactic surgeries were conducted under isoflurane anaesthesia using GCaMP6m, dLight1.1, NR1-KO and control viruses.

Cell-specific NR1-KO was performed using a CRISPR – Cas9 virus and guide RNA. The absence of the GluN1 subunit was confirmed by sequencing after fluorescence-activated cell sorting and by a lack of NMDA currents in infected cells.

In vivo stimulation of dopamine neurons was performed using AAV5-CamKII-ChR2(H134R)-eYFP injected bilaterally at +1.9 AP, 0.3 ML and 2.0 DV (mPFC).

Fibre photometry

Fibre photometry experiments were performed as before25. Mice were habituated to the testing room, handling and i.p. injections for five days before testing.

For VTA GABA neuron inhibition in the absence of drugs, an orange laser (593 nm, 10 – 15 mW) was turned on for 10 s every 1 min for 30 stimuli, and for VTA GABA neuron inhibition in the presence of ketamine, five 10-s laser stimuli were delivered.

Fluorescent indicators were excited from two excitation sources, 470 nm wavelength and 405 nm wavelength LED light, and were connected to an optic fibre patch cable and a chronically implanted optic fibre. Fibre photometry data were collected and analysed using TDT Synapse v.84.

In vivo stimulation of dopamine neurons

Mice were tethered to an optic fibre in their home cage for 1 h and received laser stimulation for 0, 15 or 60 min.

Patch-clamp electrophysiology

Patch-clamp recordings were made from mouse brain slices in artificial cerebrospinal fluid (aCSF), and dopamine neurons were recorded in a cutting solution as described previously49. Currents were amplified, filtered at 2.2 kHz and digitized at 20 kHz, and data were collected with Igor 7.

For AMPA/NMDA ratios, AMPAR currents were recorded at +40 mV holding potential without and with 2-amino-5-phosphonopentanoic acid (AP-5; 50 mM) and NMDAR currents were computed from the combined AMPAR – NMDAR trace.

NAc slices from wild-type mice were incubated in Mg2+-free aCSF with or without 50 M ketamine for 20 – 30 min, then recordings were made with high-frequency stimulation.

AAV1-FLEX-EGFP-KASH was co-injected with AAV1-CMV-FLEX-SaCas9-U6-sgGrin1 to visualize GABA neurons and to perform whole-cell recordings on EGFP-positive GABA neurons.

FOS staining and immunohistochemistry

Drd1-Tomato mice were habituated to handling and i.p. injections daily for seven days before the experiment. They were transcardially perfused with ketamine, cocaine or saline 90 min after the injections and brain sections were stained for FOS expression using standard immunohistochemistry.

Visualization of fibre location and protein expression

After fibre photometry experiments, mice were deeply anaesthetized with pentobarbital and transcardially perfused with 4% paraformaldehyde. Fixed brains were cut in 50-m-thick slices and mounted on microscopy slides with mounting medium containing DAPI.

Statistics and reproducibility

Data were analysed with Microsoft Excel 16.16.26 and GraphPad Prism 9. Mice were randomly assigned to treatment conditions using an design of interleaved trials, and experiments were replicated at least twice.