Optogenetic brain-stimulation reward: A new procedure to re-evaluate the rewarding versus aversive effects of cannabinoids in dopamine transporter-Cre mice

Abstract

Despite extensive research, the rewarding effects of cannabinoids are still debated. Here, we used a newly established animal procedure called optogenetic intracranial self-stimulation (ICSS) (oICSS) to re-examine the abuse potential of cannabinoids in mice. A specific adeno-associated viral vector carrying a channelrhodopsin gene was microinjected into the ventral tegmental area (VTA) to express light-sensitive channelrhodopsin in dopamine (DA) neurons of transgenic dopamine transporter (DAT)-Cre mice. Optogenetic stimulation of VTA DA neurons was highly reinforcing and produced a classical "sigmoidal"-shaped stimulation-response curve dependent upon the laser pulse frequency. Systemic administration of cocaine dose-dependently enhanced oICSS and shifted stimulation-response curves upward, in a way similar to previously observed effects of cocaine on electrical ICSS. In contrast, Δ⁹ -tetrahydrocannabinol (Δ⁹ -THC), but not cannabidiol, dose-dependently decreased oICSS responding and shifted oICSS curves downward. WIN55,212-2 and ACEA, two synthetic cannabinoids often used in laboratory settings, also produced dose-dependent reductions in oICSS. We then examined several new synthetic cannabinoids, which are used recreationally. XLR-11 produced a cocaine-like increase, AM-2201 produced a Δ⁹ -THC-like reduction, while 5F-AMB had no effect on oICSS responding. Immunohistochemistry and RNAscope in situ hybridization assays indicated that CB₁ Rs are expressed mainly in VTA GABA and glutamate neurons, while CB₂ Rs are expressed mainly in VTA DA neurons. Together, these findings suggest that most cannabinoids are not reward enhancing, but rather reward attenuating or aversive in mice. Activation of CB₁ R and/or CB₂ R in different populations of neurons in the brain may underlie the observed actions.

Keywords: WIN55,212-2; brain-stimulation reward; cannabinoids; cocaine; dopamine; intracranial self-stimulation; optogenetics; ventral tegmental area; Δ9-THC.

FIGURE 1

Optogenetic intracranial self-stimulation (oICSS) experiment and the effects of cocaine and Δ⁹-tetrahydrocannabinol (Δ⁹-THC) on oICSS in DAT-Cre mice. A, A schematic diagram of the AAV-ChR2-eYFP microinjection and intracranial optical fiber implantation within the ventral tegmental area (VTA) in dopamine (DA) transporter (DAT)-Cre mice. B, Image of the setup of the oICSS experiment. C, Immunostaining of whole brain slice indicating the placement of the AAV-ChR2-EYFP expression in the VTA. D, 20× magnification of the VTA showing ChR2-EYFP expression in VTA TH-positive DA neurons. E, Representative lever responding to different frequencies of laser stimulation in a single session from a single mouse. F, Graph of the lever responding over different frequencies of laser stimulation illustrating the stimulation–response curve in male and female mice. G, Cocaine (10 mg/kg, intraperitoneal [ip]) dose-dependently shifted the oICSS curve upward when compared with vehicle control. H, Δ⁹-THC dose-dependently shifted the oICSS curve downward. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the vehicle control group

FIGURE 2

Effects of cannabinoids on optogenetic intracranial self-stimulation (oICSS) in dopamine transporter (DAT)-Cre mice. A, Cannabidiol (CBD) did not show significant effects on oICSS responding. B–D, WIN55,212-2, ACEA, and AM-2201 (respectively) dose-dependently shifted the oICSS curve downward. E, 5F-AMB did not show any significant effects at the current doses. F, XLR-11, at 1.0 mg/kg, significantly shifted the oICSS curve upward. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the vehicle control group

FIGURE 3

Impact of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and WIN55,212-2 on oral sucrose self-administration in mice. Pretreatment with Δ⁹-THC A,B, or WIN55,212-2 C,D, did not alter oral sucrose self-administration

FIGURE 4

The cellular distributions of CB₁ mRNA in the ventral tegmental area (VTA) by RNAscope in situ hybridization (ISH) assays. Triple-staining for CB1, TH, and GAD1 mRNA indicates that high densities of CB₁R mRNA (green, arrows) were not co-localized with TH mRNA in VTA DA neurons (red), but co-localized with GAD₁ mRNA in GABA neuron (orange, arrows) in Vgat-Cre mice A. Selective deletion of CB₁ receptors from GABA neurons abolished CB1 mRNA-staining in the VTA of GABA-CB₁-KO mice B. TH, Tyrosine hydroxylase; GAD1, glutamic acid decarboxylase 1; DAPI, 4′,6-diamidino-2-phenylindole, a fluorescent dye that binds to DNA as a marker of cell nuclei

FIGURE 5

CB1 mRNA expression in glutamate neurons in the ventral tegmental area (VTA) by RNAscope in situ hybridization (ISH) assays. CB₁R mRNA (green, arrows) was co-localized with VgluT2 mRNA (red) in glutamate neurons (red, arrows) in VgluT2-Cre mice A, but not in glutamate-CB1-KO mice B. CB1 mRNA was still detectable in other non-glutamate (VgluT2-negative, open arrows) neurons in the VTA in glutamate-CB1-KO mice B

FIGURE 6

CB₂R-immunostaining in different phenotypes of neurons in the ventral tegmental area (VTA), illustrating CB₂R-immunostaining in VTA TH-positive DA neurons A, but not in VTA vGluT2-positive glutamate neurons B or GAD67-positive GABA neurons C. BL, baseline responding in the absence of drug treatment

FIGURE 7

Diagram summarizing CB₁R and CB₂R expression in VTA dopaminergic (DA), glutamatergic, and GABAergic neurons. Cannabinoids may bind to CB₁R in GABAergic neurons, producing rewarding or reward-enhancing effects. Conversely, cannabinoids may also bind to CB₁R on VTA glutamate neurons or glutamatergic afferents, or to CB₂R on DA neurons, producing aversive or reward-attenuating effects. The final subjective effect depends on the balance of both opposite actions. NAc, nucleus accumbens; oICSS, optogenetic intracranial self-stimulation; VTA, ventral tegmental area