Optogenetic Stimulation of Midbrain Dopamine Neurons Produces Striatal Serotonin Release

INTRODUCTION

Optogenetics entails expressing light-driven ionotropic receptors in neurons or other excitable cells to enable spatially and temporally restricted activation or inhibition.^4–7 Gene constructs for microbial or engineered rhodopsins packaged in viruses are used to transduce brain-region-specific gene expression following local delivery. Gene expression can be targeted further using Cre recombinase under the control of cell-type-specific promotors, in combination with Cre-activated opsin constructs.⁸ Opsins produce excitatory (e.g., channelrhodopsin-2, Chrimson) or inhibitory (e.g., halorhodopsin, archaerhodopsin) effects on neural activity.^9–11 The discovery and use of opsins have enabled the identification of neural pathways involved in the modulation of behavior.^12–15

Opsin-targeted cell types do not operate autonomously. Dopamine and serotonin are examples of functionally interconnected neurotransmitter systems. For instance, while dopamine signaling is often associated with reward prediction error, serotonin transmission also plays a role in processing reward-associated information.^16–18 Moreover, while widely used therapeutics for mood disorders target the serotonin system,¹⁹ the dopamine system encodes information associated with anhedonia, a core symptom of major depressive disorder.^17,20–25 Interactions between the dopamine and serotonin systems are evident in drug mechanisms of action, e.g., cocaine, methamphetamine, and 3,4-methylenedioxymethamphetamine.^26–29 Thus, these systems act in concert to modulate subjective states.^30,31

Microdialysis is a tissue sampling technique. When combined with chemical separation and detection methods, microdialysis enables the identification and quantification of neurotransmitters, metabolites, and drugs in the extracellular space.³² Several groups including ours have optimized microdialysis to monitor brain extracellular dopamine or serotonin levels via online coupling with fast separations by high performance liquid chromatography (HPLC) and electrochemical detection in awake mice and rats.^33–39 Dopamine and serotonin can be resolved in the same dialysate samples enabling biologically relevant changes in the basal and stimulated levels of these neurotransmitters to be simultaneously monitored.^39,40

Here, we set out to determine the magnitude of extracellular dopamine release in the dorsal striatum (dSTR) upon optogenetic stimulation of midbrain dopaminergic neurons. The excitatory opsin Chrimson was expressed under the control of the dopamine transporter promoter in mice. Optical activation of dopamine neurons has been used to study dopaminergic encoding of reward and movement.^15,41 In addition to dopamine, we observed optically induced increases in the dopamine metabolite 3-methoxytyramine (3-MT) and in serotonin levels. These findings demonstrate a functional link between the dopamine and serotonin systems in the basal ganglia. They illustrate the importance of monitoring multiple neurotransmitters simultaneously. And they suggest that opsin-induced behavioral changes may not be attributable solely to the neurotransmitter system or cell type targeted by opsin expression. That is to say, while optogenetics imparts highly selective control of specific types of neurons, brain function and behavior arise from distributed and interconnected networks.

RESULTS AND DISCUSSION

Using microdialysis,^33,38,39 we quantified extracellular dopamine in a dopamine-rich projection region—the dSTR—during optical stimulation of midbrain dopamine cell bodies (Fig. 1, Fig. S1). Activation of the excitatory opsin Chrimson¹⁰ produced temporally specified increases in striatal extracellular dopamine levels (Fig. 2A). To induce dopamine release we applied 50-ms square pulses for 5 minutes. These parameters were optimized to produce reproducible neurotransmitter release detectable via microdialysis. Control mice expressing mCherry or yellow fluorescent protein (YFP) in dopamine cell bodies showed no detectable changes in dopamine upon optical stimulation (Fig. 2B).

Basal (unstimulated) dialysate dopamine levels were not statistically different in Chrimson-expressing vs. control mice (Fig. 2C; see Table S1 for detailed statistics). Basal dopamine levels for control animals were normally distributed around the mean. In contrast, basal dopamine levels for Chrimson-expressing animals were not normally distributed. Individual dopamine concentrations fell mostly below the mean, apart from three animals, one of which was an outlier. Notably, this outlier is not the same animal that is an outlier for basal serotonin levels in Chrimson-transfected mice (Fig. 4a vide infra). As such, we chose to not to exclude outliers from analysis, although exclusion would have led to a statistically significant reduction in basal dopamine levels in Chrimson-transfected vs. control mice. Stimulated dopamine overflow, quantified as area under the curve (AUC), was greater in Chrimson-expressing vs. control mice (Fig. 2D; t₁₈=3.0, P<0.01). Dopamine levels were increased by ~200 pM by optical stimulation.

In addition to dopamine, optical activation led to increases in two other chromatographic peaks (Fig. S2). We perfused a selective serotonin reuptake inhibitor (SSRI) through the dialysis membrane during intracerebral dialysis to investigate peak identity in vivo as retention times commonly shift between standards and brain dialysate samples. Responses to serotonin transporter inhibition identified the smaller, later eluting peak (peak 3) as serotonin (Fig. 3A, B).

Previous experience analyzing striatal tissue samples led us to hypothesize that the remaining optically responsive peak was 3-methoxytyramine (3-MT). Dopamine is metabolized by catechol-O-methyltransferase (COMT) to produce 3-MT, which is hypothesized to function as a neuromodulator.^42,43 We administered the COMT inhibitor tolcapone systemically⁴⁴ and found that peak 2 was selectively decreased (Fig. 3C; t₃=9.6, P<0.01). We also perfused 3-MT through the dialysis membrane into dSTR, i.e., in vivo standard addition, and observed a retention time match confirming the identity of peak 2 as 3-MT (Fig. 3D).

Having identified two optically (i.e., biologically) responsive neurochemicals, in addition to dopamine, we quantified their basal dialysate levels. We found no differences in basal 3-MT or serotonin levels in Chrimson-expressing vs. control mice (Fig. 4A). Optogenetic stimulation of midbrain dopaminergic neurons evoked reproducible increases in 3-MT and serotonin in Chrimson-expressing but not control mice (Fig. 4B,C; t₁₈=3.1, P<0.01, t₁₅=4.4, P<0.001, respectively). Since basal neurochemical levels varied across individual mice (Fig. 2C, 4A), we also analyzed basal and optically stimulated neurochemical levels normalized to mean basal levels determined in control mice (Fig. S3). Concentration and %basal analyses similarly indicated that in addition to dopamine, 3-MT and serotonin overflow were increased in response to optogenetic stimulation of Chrimson-transfected dopamine neurons. Optical stimulation of control mice lacking opsin expression indicates there were no nonspecific increases in neurochemicals due to light-induced arousal and that increases in serotonin and 3-MT were due to optical activation of Chrimson expressed by dopamine neurons.

We hypothesized parsimoniously that the increased overflow of serotonin associated with optical stimulation of dopamine neurons was mediated by activation of striatal dopamine receptors on serotonin terminals. The mRNA for DRD2 and DRD3 receptors (i.e., D2-like) was previously identified in dorsal raphe.^45,46 Ren et al.⁴⁷ and Spaethling et al.⁴⁸ used single-cell transcriptomics to localize Drd2 transcripts to serotonergic neurons. Using RNAseq, Dymecki and colleagues identified Drd2 mRNA in dorsal raphe serotonin neurons specified by Pet1 expression.⁴⁹

A small number of DRN serotonin neurons has also been reported to contain Drd1a mRNA.^47,49 We carried out in situ hybridization to investigate colocalization of D1 receptor (Drd1) and serotonin transporter (Sert) mRNA in the dorsal raphe nucleus (Fig. 5A,B). We included a probe for the vesicular glutamate transporter type 3 (VGLUT3) because a subpopulation of serotonergic neurons co-expresses VGLUT3⁵⁰ and projects to the striatum.^50,51 We found that ~25% of total Sert-positive cells in the dorsal raphe were positive for Sert mRNA alone (Fig. 5C). Approximately 10% of total Sert-positive cells showed colocalization of Sert and Drd1 mRNA, while an additional 35% of SERT-positive cells showed Drd1 and Vglut3 mRNA colocalization. Positive and negative in situ hybridization controls are shown in Figure S4.

Since our data suggest that almost half of dorsal raphe serotonin neurons may express heterologous D1 receptors, we investigated whether blocking striatal D1-like receptors prevents optically stimulated serotonin overflow. We perfused SCH 23390, a D1-like receptor antagonist, into the dSTR. Basal dopamine (t₃=4.4, P<0.05) and serotonin (t₃=4.4, P<0.05) levels were increased by local D1-like receptor inhibition (Fig. 6A,B). Stimulated dopamine (t₃=6.2, P<0.01), 3-MT (t₃=4.8, P<0.05), and serotonin (t₃=4.5, P<0.05) levels were also increased by local perfusion of SCH 23390 (Fig. 6B,C). Elevations in striatal dopamine levels in response to SCH-23390 have been previously reported.⁵² In addition to serotonin neurons, medium spiny neurons (MSNs) in striatum express D1 receptors. Blocking D1-receptors on MSNs disinhibits dopamine neurons causing an increase in dopamine levels.^53,54

To focus on optically stimulated neurochemical levels, we normalized the SCH 23390 time-course data to pre-drug basal neurochemical levels determined in each mouse (Fig. 7). Similar to the dialysate concentration analysis (Fig. 6), %basal analysis indicated that SCH 23390 increased basal dopamine (t₃=4.4, P<0.05) and serotonin (t₃=4.4, P<0.05) (Fig. 7A,B). However, when normalized to respective pre-drug basal levels, potentiation of optically stimulated dopamine, 3-MT, or serotonin was no longer evident during striatal SCH 23390 perfusion (Fig. 7B,C). Thus, increases in the stimulated AUCs calculated using dialysate concentrations (Fig. 6C) were largely the result of SCH 29930-induced increases in basal dialysate concentrations.

The D1-like receptor inhibitory increase in basal serotonin levels can be explained by a circuit connecting dSTR to the DRN.¹ Approximately, 95% of projections from the dSTR to the DRN are D1-expressing MSNs,¹ which tonically inhibit DRN (and presumably serotoninergic) neurons. Blocking D1-like receptors on MSNs dendritic spines⁵⁵ could reduce tonic inhibition of DRN cell populations leading to increased serotonin levels in the dSTR. Regardless, local inhibition of D1 heteroreceptors on serotonin terminals and/or MSNs did not prevent optically evoked striatal serotonin.

Mice that received the D1-like inhibitor on day 1 of microdialysis were perfused with eticlopride (ETC), a D2-like receptor antagonist, on day 2 (Fig. 1A). Inhibition of D2-like receptors, which are expressed as dopaminergic heteroreceptors and autoreceptors in striatum,⁵⁶ was not associated with changes in basal levels of dopamine, 3-MT, and serotonin (Fig. 8A,9A). However, eticlopride perfusion into the dSTR potentiated optically evoked dopamine and 3-MT analyzed either as basal (nM) concentrations (Fig. 8B,C) or %basal levels (Fig. 9B,C), though these changes were not always statistically significant due to the small sample sizes. Previous studies have shown that extracellular dopamine is increased upon inhibition of presynaptic D₂ receptors.^57,58 Significant differences in optically evoked serotonin levels in the presence vs. absence of eticlopride were not apparent by either analysis method (Fig. 8C,9C). In the context of our current hypothesis, and like striatal D1-like receptor inhibition, D2-like inhibition did not block serotonin overflow associated with optically evoked dopamine release.

Functional interactions between the dopamine and serotonin systems have been investigated for more than 50 years.^59–61 In prefrontal cortex, dopamine receptor activation by the nonselective agonist apomorphine or local dopamine perfusion, or D2 autoreceptor inhibition by haloperidol each produced increases in serotonin levels in rats.⁶² Systemic administration of apomorphine was also shown to increase extracellular serotonin in striatum and hippocampus.⁶³ Our findings indicate that optogenetic activation of midbrain dopamine neurons expressing the excitatory opsin Chrimson produces temporally specified increases in striatal serotonin, as well as an active dopamine metabolite, 3-MT.

We tested hypotheses linking striatal dopamine and serotonin based on the idea that these neurotransmitters are released from different terminals in striatum. We found that serotonin overflow was not prevented by inhibition of striatal D1-like or D2-like receptors (Figs. 6–9). Our findings suggest that optically evoked dopamine does not produce serotonin release by stimulating dopamine receptors on striatal serotonin terminals (or direct/indirect pathway MSNs).

Our findings contrast with those of Jacobs and coworkers where apomorphine-induced or behaviorally evoked increases in striatal extracellular serotonin were inhibited by systemic and intrastriatal D2-like receptor inhibition.^63,64 Differences in species (rats vs. mice), drug (raclopride vs. eticlopride) and/or perfusion concentration (10 μM vs. 100 μM) might account for the discrepancies between studies. Jacobs and colleagues did not report on striatal dopamine levels in their studies, i.e., apomorphine and the tail-pinch and light-dark-transition behaviors may have direct receptor/serotonin system effects that are different from those mediated by evoked dopamine.⁶⁵ Artigas and colleagues reported that reverse dialysis of D1-like or D2-like agonists into striatum in rats did not alter serotonin levels supporting the idea that dopamine-serotonin interactions are not mediated by striatal dopamine receptors.⁴⁰

Another possibility is that serotonin is released from dopaminergic terminals via co-transmission or co-release. Co-transmission involves release of different neurotransmitters from different vesicle populations within the same neurons; co-release entails release of two or more neurotransmitters from the same vesicles.⁶⁶ Anatomical, genetic, and functional evidence shows that neurons can have mixed neurochemical phenotypes (for review see^66–69 among others) and argues specifically for a serotonin/glutamate mixed phenotype.^50,51

Regarding serotonin/dopamine interactions, under conditions where serotonin transporters are genetically or pharmacologically inactivated, serotonin appears to be taken up by dopamine transporters into dopamine neurons, indicated by double serotonin/tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta and ventral tegmental area.⁷⁰ Thus, SSRI treatment may result in serotonin being used as a ‘false’ transmitter by dopamine neurons. Studies on chronic SSRI administration and in vivo neurochemical monitoring are needed to test this hypothesis further. In any case, mice with wildtype serotonin transporter expression did not show serotonin colocalization in midbrain dopamine neurons suggesting that under typical circumstances, such as those investigated here, evidence is lacking for serotonin co-transmission or co-release by dopaminergic neurons.⁷⁰

Beyond striatum, a dopaminergic pathway connects the substantia nigra to the dorsal raphe, which contains a majority of forebrain-projecting serotonin cell bodies (Fig. 10). Mesostriatal serotonergic afferents project from the dorsal raphe to the striatum.² In addition to striatum, optical activation of dopamine neurons could increase extracellular dopamine in the vicinity of midbrain dopamine cell bodies. Substantia nigra dopamine neurons exhibit activity-dependent somatodendritic dopamine release and D2-mediated autoinhibition.^3,71–73 Activation of nigral D2 autoreceptors might increase extracellular serotonin in the dorsal raphe via disinhibition.⁷⁴ Furthermore, optical stimulation of dopamine cell bodies could activate dopamine projections to the dorsal raphe (Fig. 10). Both scenarios produce dopamine interactions with dorsal raphe serotonin neurons and ostensibly, could increase release of serotonin in striatum (and other brain regions).

Alternately, indirect mechanisms involving SNr-thalamus-cortex-dSTR and/or SNr-thalamus-cortex-DRN pathways cannot be ruled out.^75–78 Moreover, recent reports describe the presence of dopamine neurons in the rostral dorsal raphe nucleus.^79,80 Future experiments to parse out specific contributions from the SNr, SNc, VTA and DRN to dopamine-induced serotonin release will be informative. It is also possible that optical stimulation of dopamine neurons in Chrimson-transfected mice, in addition to releasing dopamine, is interoceptively detected by mice.⁸¹ The perception, increased arousal, and/or reward associated with dopaminergic activity could lead to increases in extracellular serotonin by complex mechanisms not involving direct connections between the dopamine and serotonin systems.

Regardless of mechanism, the present findings indicate that optogenetic stimulation of midbrain dopamine neurons evokes striatal serotonin release. We recently reported similar findings elucidated by rapid-pulse voltammetry coupled with partial least squares regression analysis.⁸² Dopamine-serotonin coupling is likely to be of importance to the facilitation of reward prediction, locomotor control, habit formation, and anhedonia.

METHODS

Supplementary Material