Striatal cholinergic interneurons and D2 receptor-expressing GABAergic medium spiny neurons regulate tardive dyskinesia

Abstract

Tardive dyskinesia (TD) is a drug-induced movement disorder that arises with antipsychotics. These drugs are the mainstay of treatment for schizophrenia and bipolar disorder, and are also prescribed for major depression, autism, attention deficit hyperactivity, obsessive compulsive and post-traumatic stress disorder. There is thus a need for therapies to reduce TD. The present studies and our previous work show that nicotine administration decreases haloperidol-induced vacuous chewing movements (VCMs) in rodent TD models, suggesting a role for the nicotinic cholinergic system. Extensive studies also show that D2 dopamine receptors are critical to TD. However, the precise involvement of striatal cholinergic interneurons and D2 medium spiny neurons (MSNs) in TD is uncertain. To elucidate their role, we used optogenetics with a focus on the striatum because of its close links to TD. Optical stimulation of striatal cholinergic interneurons using cholineacetyltransferase (ChAT)-Cre mice expressing channelrhodopsin2-eYFP decreased haloperidol-induced VCMs (~50%), with no effect in control-eYFP mice. Activation of striatal D2 MSNs using Adora2a-Cre mice expressing channelrhodopsin2-eYFP also diminished antipsychotic-induced VCMs, with no change in control-eYFP mice. In both ChAT-Cre and Adora2a-Cre mice, stimulation or mecamylamine alone similarly decreased VCMs with no further decline with combined treatment, suggesting nAChRs are involved. Striatal D2 MSN activation in haloperidol-treated Adora2a-Cre mice increased c-Fos+ D2 MSNs and decreased c-Fos+ non-D2 MSNs, suggesting a role for c-Fos. These studies provide the first evidence that optogenetic stimulation of striatal cholinergic interneurons and GABAergic MSNs modulates VCMs, and thus possibly TD. Moreover, they suggest nicotinic receptor drugs may reduce antipsychotic-induced TD.

Keywords: Cholinergic interneurons; D2 receptor-expressing neurons; GABAergic medium spiny neurons; Striatum; Tardive dyskinesia.

Conflict of interest statement

Relevant conflict of interest There are no conflicts of interest.

Copyright © 2016 Elsevier Inc. All rights reserved.

Figures

Fig. 1

Nicotine treatment reduces TD in mice. A. Treatment timeline of control/haloperidol and saccharin/nicotine treatment in mice. Haloperidol was administered for 16 wk with haloperidol-induced VCMs rated for 5 min once weekly. Chronic haloperidol increased VCMs, whether administered via daily injection (B) or via a 90-day release implantable pellet (C). Nicotine treatment reduced haloperidol-induced VCMs with both modes of haloperidol administration (B, C) with a significant effect by 11 wk of nicotine dosing. Two wk of nicotine removal increased haloperidol-induced VCMs to saccharin-treated levels (B, C right panels). Data are expressed as mean ± SEM of 5–7 mice per group. Significance of difference from own control group: #p ≤ 0.05, ##p ≤ 0.01, ### p ≤ 0.001; significance of difference from haloperidol-treated saccharin group *p ≤ 0.05, **p ≤ 0.01 ***p ≤ 0.001 using two-way ANOVA followed by a Bonferroni post hoc test.

Fig. 2

Optical stimulation of striatal cholinergic interneurons reduces haloperidol-induced VCMs via a nAChR-mediated mechanism. (A) ChAT-Cre mice were bilaterally injected with virus expressing ChR2-eYFP or control-eYFP into the striatum, and optic fiber placement done as in the timeline. They were then administered haloperidol via subcutaneously implanted slow-release pellets and VCM ratings initiated 4 wk later in the absence (No stim) and presence of stimulation (Stim). (B) Both single pulses (2 ms, 5 ms, 1s) for 5 min (left panels) and burst stimulation (5 p @ 10 Hz or 5 p @ 20 Hz) for 5 min (right panels) decreased haloperidol-induced VCMs. There was no decrease in VCMs with stimulation in mice expressing control-eYFP. (C) Haloperidol-treated ChAT-Cre expressing ChR2-eYFP or control-eYFP were rated for VCMs with and without optical stimulation (5 ms single pulse for 5 min). They were then injected with mecamylamine (1.0 mg/kg). Thirty min later, they were again rated for VCMs with (Stim) and without (No stim) optical stimulation. Stimulation or mecamylamine alone decreased VCMs to a similar extent, with no further decline in VCMs with combined stimulation and mecamylamine (Meca) treatment (left panel), suggesting the effect of stimulation is nAChR-mediated. Each bar is the mean ± SEM of 4–8 mice. In mice expressing control-eYFP there was a significant main effect of mecamylamine treatment (+p ≤ 0.05) but no effect of optical stimulation (right panel). Significantly different from No stim, ***p ≤ 0.001 using a paired t-test. Significantly different from No stim No meca, ##p ≤ 0.01 using two-way ANOVA followed by a Bonferroni post hoc test.

Fig. 3

Optical stimulation of striatal D2 MSNs reduces haloperidol-induced VCMs via a nAChR-mediated mechanism. (A) Timeline. Adora2a-Cre mice were bilaterally injected with virus expressing ChR2-eYFP or control-eYFP into the striatum, and optic fiber placement done. Haloperidol was subsequently administered via a 90-day release implantable pellet. Four wk later, the mice were rated for VCMs in the absence (No stim) and presence of stimulation (Stim). (B) Declines in VCMs were observed with single pulses of 1 s for 5 min and burst stimulation paradigms (5 p @ 10 Hz, 5 p @ 20 Hz or 25 p @ 20 Hz) for 5 min. Stimulation did not decrease VCMs in mice expressing control-eYFP. (C) Haloperidol-treated Adora2a-Cre expressing ChR2-eYFP or control-eYFP were rated for VCMs with and without optical stimulation (1 s single pulse for 5 min). They were then injected with mecamylamine (1.0 mg/kg). Thirty min later, they were again rated for VCMs with and without optical stimulation. Stimulation or mecamylamine (Meca) alone decreased VCMs to a similar extent, with no further decline in VCMs with combined stimulation and mecamylamine treatment (left panel), suggesting the effect of stimulation occurs via nAChRs. In mice expressing control-eYFP there was a significant main effect of mecamylamine (++p ≤ 0.01) but no effect of optical stimulation (right panel). Each bar is the mean ± SEM of 4–8 mice. Significantly different from No stim, *p ≤ 0.05, **p ≤ 0.01 using a paired t-test. Significantly different from No stim No meca, #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.001 using two-way ANOVA followed by a Bonferroni post hoc test.

Fig. 4

Distribution of ChR2-eYFP in striatum of transgenic mice. (A) Expression of ChR2-eYFP (green) in the right striatum (A1.4 to A0.2) of ChAT-Cre mice. (B) The boxed area in the schematics are depicted in the images in (A) and (C). (C) Expression of ChR2-eYFP (green) in the right striatum (A1.4 to A0.2) of D2 MSNs (Adora2a-Cre mice). Scale bar = 500 µm. Ctx, cortex; Str, striatum. (D) Expression of ChR2-eYFP in striatal cholinergic interneurons of ChAT-Cre mice and (E) expression of ChR2-eYFP in striatal MSNs of Adora2a-Cre mice. Images depict localization of ChAT (red, left panel D) or DARPP-32 (red, left panel E) immunofluoresence, ChR2-eYFP (green, middle panel) and the merge (yellow, right panel). Scale bar = 20 µm.

Fig. 5

Activation of striatal D2 expressing GABAergic MSNs in haloperidol-treated Adora2a-Cre mice increases c-Fos+ D2 MSNs and decreases c-Fos+ non-D2 MSNs. Adora2a-Cre mice expressing ChR2-eYFP or control-eYFP were implanted with haloperidol pellets to induce TD. They were then optically stimulated (ChR2-eYFP Stim) with single pulses (1 s) for 5 min, a condition that reduces TD. There was also a subset of ChR2-eYFP mice that received haloperidol but were not optically stimulated (ChR2-eYFP No stim). The mice were then killed immediately after stimulation, the brain quickly removed and fixed for immunofluorescence. A, depicts neurons in the boxed area in B showing cells immunostained with ChR2-eYFP (green, left), c-Fos (red, middle) and the merge (right). B, provides images of eYFP (green), c-Fos (red) and eYFP+c-Fos (yellow) positive neurons of unstimulated ChR2-eYFP mice (No stim, left), ChR2-eYFP mice with stimulation (Stim, middle) and control-eYFP mice with stimulation (Stim, right). Quantitation of the data is provided in C, with the boxed area in the image depicting the dorsolateral striatal area counted. D and E, respectively, depict images and quantitation of the effect of cholinergic neuron activation in haloperidol-treated ChAT-Cre mice; no changes were observed in c-Fos+ ChAT neurons with stimulation. Each bar is the mean ± SEM of n = 4–5 ChR2-eYFP (No stim), n = 4 ChR2-eYFP (Stim) and n = 3 Control-eYFP (Stim) mice. Significantly different from ChR2-eYFP (No stim), *p ≤ 0.05, **p ≤ 0.01; from Control-eYFP (Stim), +p ≤ 0.05 using a one-way ANOVA followed by a Dunnett’s post hoc test. Scale bar = 20 µm and 100 µm in the upper and middle panel, respectively.