Introduction

Dopamine projections to the striatum underlie many important functions, including movement control and reinforcement learning (Schultz, 2002). Loss of dopamine innervation to the dorsal striatum leads to Parkinson's disease, and excessive dopamine in the same region can lead to the positive symptoms of schizophrenia (Gerfen and Engber, 1992; Chen et al., 2024). Furthermore, the actions of dopamine in the striatum are implicated in substance use disorder and other compulsive behaviors (Nestler, 2005; Jalal et al., 2023). Understanding dopamine release regulation in the striatum will provide mechanistic knowledge of these physiological and pathological states.

Much of what we know about dopamine release in the striatum, particularly at the synaptic level, comes from work in rodents (Rice et al., 2011; Gantz et al., 2018; Ozcete et al., 2024). Mice are an important model system because of the availability of genetically modified lines which, in combination with virally delivered constructs, provide detailed and precise control over function at the molecular, synaptic, and cellular levels. While mice offer unique advantages, there are important differences in forebrain circuits between mice and primates which make it unlikely that dopamine release and its regulation would be the same across the species. These differences encompass the overall scale of the forebrain and the existence of several structures in primates that are not found in mice, such as the granular lateral prefrontal cortex, the internal capsule, and a well-defined globus pallidus internal segment (https://scalablebrainatlas.incf.org/macaque/PHT00; Bezgin et al., 2009; Bakker et al., 2015). Relevant for dopamine regulation, there are differences between rodents and primates in the organization of the reciprocal interaction between the striatum and midbrain dopamine neurons (Joel and Weiner, 2000). For example, in rodents the projections from the motor and associative striatum to the dopamine neurons are largely topographically segregated into a lateral-medial gradient in the midbrain while in primates caudate and putamen projections overlap in the midbrain, suggesting convergence. Similarly, the dopamine neuron populations that project to lateral and medial striatal subregions are topographically segregated in rodents while in primates they form interdigitated clusters. The dopamine innervation of the cortex is also expanded in primates relative to rats and shows differences in the laminar pattern of projections (Berger et al., 1991). This suggests that dopamine release and regulation may be different between rodents and primates. In fact, the comparison of evoked dopamine release in the striatum between guinea pigs and marmosets, a new-world primate, has shown increased dopamine release in the marmoset relative to the guinea pig (Cragg et al., 2000). However, a study comparing mice and macaques found the opposite, with macaques showing less striatal dopamine release than mice (Calipari et al., 2012). Understanding the comparative similarities and differences between rodent and primate dopamine systems can lead to a better understanding of the ways in which findings in rodents translates, ultimately, to humans.

The striatum, a nucleus dominated by GABA-expressing neurons, relies on inhibition and disinhibition as critical features of the local circuitry that drive computation and information processing. Among the multiple interspecies differences between rodents and primates, an expansion of inhibitory cell types has been shown in primate cortex and striatum (Boldog et al., 2018; Krienen et al., 2020; Corrigan et al., 2024), raising the possibility that inhibition and disinhibition are different in primate brains. In mice, GABA receptors are known to influence dopamine release through multiple mechanisms, including by sensing striatal GABAergic tone and GABA coreleased from dopamine axons themselves (Gruen et al., 1992; Smolders et al., 1995; Pitman et al., 2014; Lopes et al., 2019; Kramer et al., 2020; Holly et al., 2021; Patel et al., 2024). Based on the reported difference between species in proportion of GABA-expressing neurons, we speculated that GABA modulation of dopamine transmission might be changed in macaques compared with mice.

In addition to GABA, striatal dopamine release is also modulated by acetylcholine. Acetylcholine regulates dopamine release via nicotinic receptors expressed on dopamine axons within the striatum (Jones et al., 2001; Rice et al., 2011; Kramer et al., 2022). This nicotinic-mediated regulation of dopamine release may serve specific functional roles that are distinct from release evoked by the firing of dopamine neurons initiated at the soma (Mohebi et al., 2019; Adrover et al., 2020; Kramer et al., 2022; Liu et al., 2022). For example, whereas spiking activity in dopamine neuron cell bodies correlates with reward prediction errors under many conditions (Fritsche et al., 2024; Schultz, 2024), regulation of terminal release within the striatum might correlate with state value and/or salience (Hamid et al., 2016; Mohebi et al., 2019; Jeong et al., 2022; Gershman et al., 2024).

In the present study, we compared dopamine release capacity and regulation between mouse and macaque ex vivo brain slices. Earlier work on this topic relied on fast-scan cyclic voltammetry (FSCV), an electrochemical technique. Here, in addition to voltammetry, we further approached this question of interspecies comparison with optical methods using virally expressed genetically encoded fluorescent dopamine sensors, dLight1.3b and GRAB-DA1m. Furthermore, we examined the regulation of dopamine release by two neurotransmitters known to influence release in rodents: acetylcholine and GABA. In agreement with previous work, we found smaller evoked dopamine signals in macaques compared with mice and the existence of a ventrodorsal gradient in both species, with larger dopamine release in the putamen/dorsolateral striatum than the ventral striatum/nucleus accumbens. Extending from the previous report, this study identifies that the cholinergic contribution to the evoked dopamine release is smaller in macaques compared with mice, possibly explaining the smaller overall amplitude in macaques. Indeed, removing the cholinergic contribution led to evoked dopamine levels that were comparable between mice and macaques. Finally, dopamine regulation by GABA was stronger in macaque than mouse brain slices. Blocking both ionotropic and metabotropic GABA receptors produced a stronger potentiation of dopamine release in the macaque striatum compared with the mouse striatum. Altogether, this comparative study offers evidence of evolutionary divergence on dopamine release regulation between mice and macaques.

Results

In these experiments, we compared electrically evoked dopamine release across different subregions of the striatum, and between mice and macaques. Evoked dopamine was measured in ex vivo brain slices using both FSCV and, in some cases, the dopamine-sensitive fluorescent reporters dLight1.3b and GRAB-DA1m. We further used pharmacological methods to examine the relative contributions of cholinergic and GABA-mediated mechanisms to the evoked dopamine release.

Coronal slices were taken from rhesus macaques (n = 17 animals; 7 males/10 females) and mice (n = 45 animals; 31 males/14 females). Recordings were carried out in specific subregions of the striatum. In the macaque, subregions were identified by their location relative to the internal capsule. In mice, they were identified by their gross anatomical position (Fig. 1A). In the monkey we identified the caudate, putamen, and nucleus accumbens (NAc); in the mouse we recorded from the dorsomedial (DMS), the dorsolateral striatum (DLS), and the NAc (Heilbronner et al., 2016). NAc recordings were made from core and shell areas but data was not segregated due to smaller sample size. All experimental protocols were the same across species and recordings were carried out on the same slice rigs using the same equipment and solutions.

Figure 1.

Dopamine signals across the striatum are smaller in macaques compared with mice. A, Coronal section images from macaque (left) and mouse (right) delineating the striatal subregions recorded. Images adapted from Scalable Brain Atlas. NAc, nucleus accumbens; DMS, dorsomedial striatum; DLS, dorsolateral striatum. B, C, Superimposed representative dopamine transients evoked by a single-pulse electrical stimulation of increasing intensity (black tick) from (B) the putamen of monkey or (C) the DLS of mouse. D, The input–output curves show mean peak amplitude of dopamine signals evoked at each stimulation intensity in the mouse (blue) and macaque (red) striatal subregions (n = 33, 23, 31 slices/12, 6, 4 mice; n = 23, 31, 16 slices/19, 10, 5 macaques). Symbols and lines are mean ± SEM.

We first examined electrically evoked dopamine release with voltammetry using a series of stimulation currents. Across both species and all striatal subregions, increased stimulation intensity led to increased peak concentration of evoked dopamine signals (Fig. 1B–D; three-way repeated-measures ANOVA main effect of stimulation: F(7,1066) = 220.1, p < 0.001), but with significant differences across striatal subregions (Fig. 1D; main effect of area: F(2,148) = 9.2, p < 0.001). In both species, post hoc analyses showed that the NAc had significantly lower peak concentrations of evoked dopamine than the putamen/DLS and caudate/DMS (post hoc Sidak test both ps < 0.001). However, the putamen/DLS and the caudate/DMS did not differ statistically within each species (post hoc Sidak test ps > 0.07). Though both species exhibited similar subregion differential response profiles (Fig. 1D; interaction species × area: F(2,148) = 2.0, p = 0.138), evoked dopamine release was lower across all subregions in monkeys compared with that in mice (main effect of species: F(1,148) = 105.9, p < 0.001). Thus, macaques showed overall lower peak concentrations of evoked dopamine release (400 vs ~800 nM), and dopamine levels were overall lower in the NAc in both species (0.09 ± 0.02 µM in macaques vs 0.59 ± 0.08 µM in mice).

Next, to further validate these voltammetry results, we examined evoked dopamine release using genetically encoded dopamine sensors coupled with photometry. These sensors, consisting of modified dopamine receptors, are expressed directly on the striatal cell membrane, allowing for measurements of dopamine concentrations that more accurately reflect extracellular levels sensed by neurons near dopamine axon boutons (Dong et al., 2022). This is in contrast to the carbon fiber electrode used in voltammetry, which is inserted into the tissue and primarily measures dopamine spillover from release sites. Viral vectors expressing the sensors were injected in the putamen/DLS, and brain sections were prepared 6–8 weeks later to ensure adequate levels of sensor expression (Fig. 2A–C). Critically, this independent measure of dopamine concentration confirmed that evoked dopamine transients were larger in the mouse than macaque, as measured by both dLight1.3b (Fig. 2D; main effect of species F(1,28) = 27.9, p < 0.001) and GRAB-DA1m (Fig. 2E; main effect of species F(1,25) = 57.2, p < 0.001). In some experiments we carried out simultaneous voltammetry and photometry (Fig. 2F). This allowed us to ask whether the presence of the dopamine sensor, which binds dopamine, affects dopamine levels measured with voltammetry. Interestingly, we found that the expression of dopamine sensors had an effect on the amplitude of the dopamine response measured with voltammetry in macaques. Across the stimulation intensities tested, the evoked dopamine responses measured with voltammetry in regions expressing the dopamine sensors were smaller (∼ half) than dopamine responses recorded in regions from the same slice but without sensor expression (main effect of sensor expression; Fig. 2F; F(1,36) = 4.9, p = 0.033). This was the case for both sensors, dLight1.3b and GRAB-DA1m. In mice, there were no differences in evoked dopamine levels from regions with or without expression of these same sensors (Fig. 2F; no effect of sensor; F(1,32) = 0.04, p = 0.839). There was, however, no significant interaction of species and sensor at the highest stimulation level (F(1,68) = 2.3, p = 0.134).

Figure 2.

Dopamine fluorescent sensors confirms smaller magnitude of evoked dopamine signals in macaques. A, B, Representative photometry traces obtained from macaque brain slices expressing the fluorescent dopamine sensors dLight1.3b (A) and GRAB-DA1m (B). Dopamine signals were evoked by increasing stimulation intensities. Top right corner shows fluorescence images of brain sections from macaque caudate and putamen. C, Input–output curves of photometry signals measured in macaque brain sections expressing dLight1.3b (filled symbols, n = 14 slices/3 macaques) and GRAB-DA1m (open symbols, n = 11 slices/ 2 macaques). D, E, Photometry input–output curves macaque striatum (red) and mouse striatum (blue) when using dLight1.3b (14 slices/3 macaques; 16 slices/7 mice) and GRAB-DA1m (slices n = 11 slices/2 macaque; 16 slices/6 mice). F, Voltammetry input–output curves from mouse (blue) and macaque (red) dorsal striatum expressing the fluorescent sensors (open symbols) or not expressing the sensors (filled symbols).

We next examined the effects of pharmacological manipulations on evoked dopamine release recorded in dorsal striatum slices. We first examined the regulation by nicotinic acetylcholine (ACh) receptors (nAChR) by comparing evoked dopamine release levels before and after application of the β2 subunit containing nAChR blocker DHβE. Across species (Fig. 3A–C), evoked dopamine levels were significantly smaller in the presence of DHβE at the highest stimulation tested (main effect of blocker F(1,33) = 118.2, p < 0.001). Pairwise comparisons found that both macaques (F(1,52) = 5.9, p = 0.019) and mice (F(1,14) = 16.3, p = 0.012) had significantly smaller dopamine responses following DHβE. There was also a significant interaction between species and drug condition (F(1,33) = 31.6, p < 0.001), suggesting the effects of nAChR were different. Indeed, the magnitude of the DHβE suppression was smaller in macaques than in mice (41 ± 3 vs 64 ± 5% decrease in mice).

Figure 3.

Smaller cholinergic contribution to evoked dopamine signals in macaques compared with mice. A, Representative dopamine transients evoked by single-pulse electrical stimulation (tick) before (pale) and after 1 µM DHβE application (solid) in macaque (red) and mouse (blue). B, C, The input–output curves of dopamine amplitudes with increasing stimulation intensities in macaque (B) and mouse (C) before (pale) and after (solid) 1 µM DHβE bath application. n = 27 slices/4 macaques, n = 26 slices/9 mice. D, Proportion of dopamine peak amplitudes blocked by the nicotinic receptor blocker DHβE in macaque (red) and mouse (blue) dorsal striatum from male (white) and female (orange) animals. Bars are mean ± SEM, and symbols are individual values. E, Time course of dopamine peak amplitudes as 1 and 10 μM DHβE were bath-applied in macaque caudate and putamen slices. Data is mean ± SEM normalized to predrug application. F, Time course of the dopamine peak amplitudes during application of nicotinic blocker conotoxin-PIA (0.1 µM) followed by 1 µM DHβE. Data is mean ± SEM normalized to predrug application for macaque (red) and mouse (blue), n = 5 slices/2 macaque; 15/2 mice.

Calculating the fraction of evoked dopamine that was dependent versus independent of nAChR activity (Fig. 3D) revealed that the nAChR-dependent component was larger in mice than that in macaque, and correspondingly the nAChR-independent component was smaller in mice than that in monkeys (t(39) = 6.8, p < 0.001). To determine whether this species difference reflects poor sensitivity to the receptor blocker in macaques, we carried out an additional experiment in which we increased the concentration of DHβE 10-fold. However, increasing the blocker concentration to 10 µM did not further reduce evoked dopamine levels relative to the 1 µM application (Fig. 3E; t(3) = 1.14, p = 0.337). Thus, the 1 µM concentration of DHβE appears sufficient to block the existing nicotinic receptors in the macaques, but these β2-containing receptors contribute less to the overall evoked dopamine release triggered by electrical stimulation in macaques than in mice.

We further examined the subtype composition of nAChRs that contribute to dopamine release in mice and monkeys using another receptor blocker. α-Conotoxin-P1A (Ctx-PIA) is a blocker for nAChRs that contain α6 subunits (Dowell et al., 2003). Application of Ctx-PIA significantly reduced evoked dopamine signals in both mice (23 ± 1% reduction) and macaques (41 ± 1% reduction; Fig. 3F; main effect of drug F(17,323) = 38.34, p < 0.0001). However, the effect of blocking α6-containing nicotinic receptors was larger in macaques (macaque vs mouse post hoc test, t(15) = 2.76, p = 0.006). Subsequent addition of DHβE produced no additional decrease in macaque (6 ± 1% reduction, post hoc macaque Ctx-PIA vs DHβE t(5) = 0.6, p = 0.5) but further decreased dopamine signals in mice by 37% to 40 ± 3% from the predrug administration (post hoc mouse Ctx-PIA vs DHβE t(15) = 8.4, p < 0.0001). There was a significant interaction between species and pharmacology when we compared Ctx-PIA and DHβE conditions (F(17,323) = 3.4, p < 0.0001). This result indicates that α6-containing nAChRs exert the most control over dopamine release in the caudate and putamen of macaques and suggests a loss over the course of evolution of non-α6-containing receptors in macaques, potentially accounting for the diminished cholinergic-mediated dopamine release and the smaller evoked dopamine signals in monkey slices.

Over 95% of striatal neurons are GABAergic and the striatum is therefore dominated by inhibition. Thus, we next examined the effect of blocking GABA receptors on evoked dopamine release. We found that a combination of gabazine and CGP55845 (GZ/CGP), which block GABAA and GABAB receptors, respectively, increased evoked dopamine release (Fig. 4A–C). The voltammograms and current–voltage plots showed selective changes in the carbon fiber currents only at two voltages characteristic for dopamine oxidation and reduction, strongly suggesting an increase in the evoked extracellular dopamine concentration after blockers. The potentiation was seen in both species [Fig. 4A–C; main effect of blockers F(2,48) = 35.86, p < 0.0001 and species (F(1,21) = 14.42, p < 0.001)]; but it was proportionally larger in macaques than mice (Fig. 4D; main effect of species F(1,21) = 14.42, p = 0.001). We found a significant species × GABA blockers interaction (F(14,294) = 9, p < 0.0001), suggesting a stronger inhibitory tone in the primate striatum compared with mouse.

Figure 4.

Stronger inhibitory modulation by GABA over striatal dopamine signals in macaques than mice. A, Representative traces of dopamine signals (right), the current–voltage plots (left top), and the voltammograms (left middle and bottom) evoked by a single pulse of electrical stimulation (tick) in macaque (red) and mouse (blue) striatum before and after (green) bath application of the GABA receptor blockers (5 μM gabazine and 2 μM CGP55845). B, C, The input–output curves of dopamine amplitude evoked by stimulation of increasing intensity before and after (green) GABA receptor blockers in macaque (B, red) and mouse (C, blue). Symbols and lines are mean ± SEM. n = 13 slices/3 macaque; 12 slices/6 mice. D, E, Time course of the dopamine amplitudes evoked at 300 µA during application of the GABA receptor blockers when done in the (D) absence (n = 11 slices/4 macaque; 12 slices/6 mice) or (E) presence of 1 µM DHβE in macaque (red) and mouse (blue) striatum (n = 29 slices/4 macaque; 13 slices/4 mice). Amplitude is normalized to predrug application (E). Symbols and lines are mean ± SEM. F, Percent change in the dopamine peak amplitudes evoked by 300 µA after GABA receptor blockers in macaque (red) and mouse (blue) bars were plotted as bar graphs together with individual values for the two species. Bars and lines are mean ± SEM, and symbols represent data from single experiments shown in white symbols for male and orange for female. n = 13 slices/4 macaques in ACSF and 31 slices/4 macaques in DHβE; 12 slices/6 mice in ACSF and 13 slices/4 mice in DHβE.

To examine possible interactions between GABAergic- and cholinergic-mediated modulation of dopamine release, we tested the effect of GABA blockers after application of the nicotinic receptor blocker DHβE (Fig. 4E). In the presence of DHβE, GABA receptor blockers still produced a substantial increase in dopamine release in macaque but not in mice (Fig. 4E; t(39) = 6.8, p < 0.001). Blocking nicotinic receptors did not prevent the disinhibition produced by GABA receptor blockers in macaques, suggesting the mechanisms underlying the GABA-mediated modulation of release are independent from those of ACh and could be additive in the macaque. In mouse, the GABA blocker-mediated disinhibition was not observed after DHβE, suggesting the cholinergic and GABAergic modulation of dopamine release work, at least partially, through a common mechanism, in agreement with a recent report (Brill-Weil et al., 2024). However, gabazine has also been reported to decrease the sensitivity of carbon fibers to dopamine (Patel et al., 2024). In our experiments, using 5 µM gabazine led to an approximate 18 ± 10% decrease in dopamine sensitivity. In the presence of a nicotinic receptor blocker, gabazine induced a rapid suppression of evoked signals, followed by a recovery to baseline (Fig. 4E). This suggests that the initial suppression might be due to gabazine's effects on carbon fiber sensitivity, while the recovery to baseline could result from gabazine's effect on disinhibiting dopamine release.

To further assess the modulation and their interactions, we evaluated the relative proportion of GABA blocker potentiation in each species, before and after nAChR blockers (Fig. 4F). Evoked dopamine release was significantly greater across species when GABA blockers were applied without DHβE (main effect of DHβE F(1,69) = 5.5, p = 0.021). As stated before, the potentiation of evoked dopamine responses by GABA blockers is larger in macaques than that in mice (t = 2.9, p = 0.005). Furthermore, in macaques GABA receptor blockers potentiated dopamine release similarly before and after DHβE (t = 1.1, p = 0.25), suggesting that inhibition is not acting on the nicotinic component of the release mechanism in macaques. In the mouse, however, the small potentiation by GABA receptor blockers is absent after DHβE (t = 2.1, p = 0.039), suggesting that inhibition affects the cholinergic-dependent dopamine release mechanism.

Finally, we measured the overall magnitudes of the cholinergic- and GABAergic-mediated effects on regional evoked dopamine release in mice and macaque. We observed larger evoked dopamine signals in mice than macaques and regional differences in both species (Fig. 5A; main effect of region F(2,116) = 8.59, p = 0.0003 and species F(1,116) = 58.20, p < 0.0001; no interaction p = 0.32), in agreement with previous work (Calipari et al., 2012). Isolating the cholinergic-dependent dopamine release using pharmacology shows differences between striatal subregions and species (main effect of region F(2,116) = 5.87, p = 0.0037 and species F(1,116) = 95.96, p < 0.0001; no interaction p = 0.66). These cholinergic-dependent signals were smaller in macaque than those in mouse for all subregions (post hoc macaque vs mouse t's > 4.3, p's < 0.0001). We then analyzed and compared the input–output curves for the dorsomedial striatum/caudate, for which we have the most data points in both species. In the mouse, these cholinergic-dependent dopamine signals are twice the size of the remaining signals, and the input–output curve shows an upward shift with no change in sensitivity compared with the curve in macaques (Fig. 5B; significant interaction stimulation × species F(10,510) = 6.57, p < 0.0001; intensity 15–20 μA mouse vs macaque t > 3.2, p < 0.01). On the contrary, the remaining evoked dopamine signal, which is independent of nicotinic receptors, has similar amplitudes in mice and macaques (Fig. 5A, no species effect F(1,118) = 2.44, p = 0.12), and there are regional differences (F(2,118) = 8.57, p = 0.0003). The input–output curve of this nicotinic-independent component in the macaques also showed a leftward shift compared with mice and an interaction between stimulation and species (Fig. 5C; F(10,330) = 3.12, p = 0.0008), suggesting evoked dopamine signals in macaques have higher sensitivity to electrical stimulation.

Figure 5.

Shift from acetylcholine to GABA modulation of dopamine signals in macaque compared with mice. A, Bar plot shows the overall magnitude of the evoked dopamine signals for maximal stimulation (300 µA) across striatal subregions in macaque (red) and mouse (blue). Solid portion represents the amplitude remaining after DHβE, denoting cholinergic-independent release. Light portion represents the amplitude of cholinergic-dependent release, which was blocked by DHβE. n = 61 slices/13 macaque; n = 61 slices/11 mice. * significant difference from mouse subregion. NAc, nucleus accumbens; DMS, dorsomedial striatum; DLS, dorsolateral striatum. B, The input–output curves of the cholinergic-dependent dopamine signals in the macaque caudate (red) and the mouse DMS (blue). C, The input–output curves of the non-ACh-dependent dopamine signals in the macaque caudate (red) and the mouse DMS (blue). n = 27 slices/4 macaques, n = 26 slices/9 mice. D, Amplitude of evoked dopamine signals at maximal stimulation (300 µA) for macaque caudate (red) and mouse DMS (blue) before and after GABA receptor antagonists gabazine and CGP (green). * main effect of GABA blockers. n = 13 slices/3 macaque; 12 slices/6 mice. E, The input–output curves of the GABA-modulated dopamine signals in the macaque caudate (red) and the mouse DMS (blue). n = 12–13 slices/3–4 animals. F, Amplitude of evoked dopamine signals remaining after DHβE at maximal stimulation (at 300 µA) for macaque caudate (red) and mouse DMS (blue) before and after GABA receptor antagonists gabazine and CGP (green) in the presence of nicotinic receptor antagonist. # significant interaction species × GABA blocker. n = 31 slices/4 macaques; 13 slices/4 mice. For all plots, symbols and lines are mean ± SEM. G, Diagram summarizing main findings and a model for interpreting the species differences.

Evaluation of the isolated GABA-modulated component of evoked dopamine release revealed that GABA blockers produced robust potentiation (Fig. 5D) and displayed overlapping input–output curves in macaques and mice (Fig. 5E). The maximal amplitude of the GABA-modulated dopamine signals and the sensitivity to stimulation are similar in macaques and mice (no effect of species F(1,23) = 0.25, p = 0.62). However, there were two differences between the species. First, the potentiation is proportionally larger in macaques because they have smaller overall initial signals (44 ± 5% in macaques vs 19 ± 3% in mice, t = 2.93, p = 0.0046). Second, the potentiation persisted after blocking nicotinic receptors mainly in macaques (Figs. 4E,F, 5F; 36 ± 4% in macaques vs 1 ± 3% in mice, t = 5.06, p < 0.0001), which may reflect that GABA-mediated modulation is more robust in macaque than mice.

Discussion

The goal of this study was to perform a detailed characterization of ex vivo evoked dopamine signals in the striatum of macaques and mice. By using the same recording equipment and experimental conditions, our experiments allowed for a systematic comparison between the two species. We observed differences in the overall magnitude of evoked dopamine signals between species and regional variations within species, confirming and validating previous findings (Calipari et al., 2012). Our work also provides mechanistic insight by demonstrating that cholinergic-dependent dopamine release is smaller in macaques compared with mice (~40 vs ~60%) and that the tonic inhibition by GABA is larger in macaque (44 vs 19%), both of which contribute to the difference in amplitude between species.

Technical and other biological considerations on dopamine measurements

Dopamine concentration was measured using both the classical electrochemical method of FSCV and a more recent approach—photometry, which utilizes engineered dopamine receptors that increase fluorescence emission upon dopamine binding (Dong et al., 2022). Although each method comes with limitations, both techniques showed consistently lower concentrations of extracellular dopamine in macaques compared with mice. One of the limitations of the photometry measurement is that the expression level of dopamine sensors can be different between the two species, and it could affect the comparison. Lower expression level of the sensor in macaques could contribute to the disproportionately lower signals measured in macaque with photometry versus FSCV. Interestingly, in macaques, where evoked dopamine signals were smaller, the expression of dopamine sensors further reduced the dopamine concentration detected by the carbon fiber during FSCV. In mice, however, the expression of the dopamine sensors did not impact the dopamine concentration measured by FSCV.

A few other technical considerations are essential for interpreting these findings. Voltammetry has a slower detection rate compared with photometry (10 Hz vs 5 kHz) and lower sensitivity, as the carbon fiber electrode is inserted into the tissue and measures dopamine that “spills over” from release sites. In contrast, dopamine sensors like dLight1.3b and GRAB-DA1m have affinities similar to endogenous D1 and D2 receptors, respectively, and are expressed on cell membranes, allowing them to detect extracellular dopamine close to the release sites. However, due to their high ligand affinity, these sensors may exhibit a reduced effective dynamic range and potentially interfere with dopamine binding to endogenous receptors, thus affecting signaling. Our results indicate that in macaques, the expression of dopamine sensors diminished the dopamine concentration detected by FSCV, which may reflect a reduction in available extracellular dopamine for binding to endogenous receptors when the sensors are expressed. These findings highlight the importance of considering potential interference when using dopamine sensors for in vivo measurements during behavior.

The circadian influence on dopamine content and evoked striatal release should be taken into account, as it may impact each species differently. Since all experiments were conducted during the light phase of the circadian cycle, circadian variations in dopamine levels likely affect macaques and mice differently, given that mice are nocturnal. Enzymes involved in dopamine synthesis and degradation, such as monoamine oxidase A and tyrosine hydroxylase (TH), are regulated by clock genes and the circadian rhythm (Hampp et al., 2008; Sidor et al., 2015; Verwey et al., 2016). Based on these studies, we expect that TH expression and activity are at their lowest during the sleep/inactive phase of the mouse circadian cycle, which coincides with when they were tested. Therefore, while we cannot rule out the possibility that circadian factors contribute to the observed interspecies differences in evoked dopamine levels, these factors alone cannot explain the higher evoked levels observed in mice compared with macaques.

Cholinergic modulation of dopamine release

Cholinergic modulation of dopamine release is observed across all striatal subregions; however, in both species, it is enhanced in the medial and lateral dorsal striatum and less pronounced in the NAc. After blocking the cholinergic-dependent signals, the remaining evoked dopamine signals are of similar amplitudes between species, with the macaque signals exhibiting higher sensitivity to stimulation while nAChRs are blocked. Additionally, in the mouse, the cholinergic component of evoked dopamine release is dependent on both α6- and non-α6-containing nAChRs. Whereas non-α6-containing nAChRs contribute up to half of evoked dopamine release in mice, α6-containing nAChRs are responsible for nearly all cholinergic-dependent dopamine release measured in vitro in the macaque caudate and putamen (Fig. 3F). Therefore, it is possible that there is an evolutionary loss of this non-α6-containing nAChR in macaque, or specific gain in the mice, that is responsible for the diminished overall cholinergic-dependent dopamine release in the macaque striatum. Alternative explanations for the interspecies difference include nAChR density, axonal location of the receptors, strength and dynamics of presynaptic ACh release, and the density of cholinergic terminals, among others. These factors should be investigated in future studies.

GABA modulation of dopamine release

We also identified local modulation of dopamine release by the neurotransmitter GABA. GABA is abundant in the striatum, with over 95% of the cells releasing GABA (Burke et al., 2017). Furthermore, while most afferents to the striatum release glutamate, GABA release from midbrain dopamine neurons and midbrain GABAergic projection neurons has also been reported in mice (Tritsch et al., 2016). The local modulation by GABA, revealed by application of GABAA and GABAB receptor blockers, potentiated dopamine release in both macaques and mice. These findings agree with recent published work showing GABAA receptor-mediated suppression of dopamine release in mice (Kramer et al., 2020; Brill-Weil et al., 2024; Patel et al., 2024).

The absolute magnitude of the dopamine potentiation in the presence of GABA blockers was similar in macaques and mice, with no change in the sensitivity to stimulation. But since the overall evoked release in macaques was one-third of that of mice, the potentiation was proportionally larger in macaques than mice (~50 vs ~15% in mice). Inhibition and disinhibition are common features of striatal networks, aiding in the processing and decoding of information within these GABA-dominated circuits. Dopamine itself has been shown to modulate GABA release in the striatum. This is thought to be a key action of dop