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. Author manuscript; available in PMC: 2020 Dec 1.
Published in final edited form as: Eur J Neurosci. 2019 Jul 16;50(12):3968–3984. doi: 10.1111/ejn.14493

The Ventral Tegmental Area has calbindin neurons with the capability to co-release glutamate and dopamine into the nucleus accumbens

Smriti Mongia 1, Tsuyoshi Yamaguchi 1,2, Bing Liu 1, Shiliang Zhang 1, Huiling Wang 1, Marisela Morales 1
PMCID: PMC6920608  NIHMSID: NIHMS1036454  PMID: 31215698

Abstract

The ventral tegmental area (VTA) has three major classes of neurons: dopaminergic (expressing tyrosine hydroxylase; TH), GABAergic (expressing vesicular GABA transporter; VGaT) and glutamatergic (expressing vesicular glutamate transporter 2; VGluT2). While VTA dopaminergic and GABAergic neurons have been further characterized by expression of calcium-binding proteins (calbindin, CB; calretinin, CR or parvalbumin, PV), it is unclear whether these proteins are expressed in rat VTA glutamatergic neurons. Here, by a combination of in situ hybridization (for VGluT2 mRNA detection) and immunohistochemistry (for CB−, CR− or PV-detection), we found that among the total population of VGluT2 neurons, 30% coexpressed CB, 3% coexpressed PV and less than 1% coexpressed CR. Given that some VGluT2 neurons coexpress TH or VGaT, we examined whether these neurons coexpress CB, and found that about 20% of VGluT2-CB neurons coexpressed TH and about 13% coexpressed VGaT. Because VTA TH-CB neurons are known to target the nucleus accumbens (nAcc), we determined whether VGluT2-CB-TH neurons innervate nAcc, and found that about 80% of VGluT2-CB neurons innervating the nAcc shell coexpressed TH. In summary (a) CB, PV and CR are detected in subpopulations of VTA-VGluT2 neurons; (b) CB is the main calcium-binding protein present in VTA-VGluT2 neurons; (c) one-third of VTA-VGluT2 neurons coexpresses CB; (d) some VTA-VGluT2-CB neurons have the capability to co-release dopamine or GABA, and (e) a subpopulation of VTA-glutamatergic-dopaminergic neurons innervates nAcc shell. These findings further provide evidence for molecular diversity among VTA-VGluT2 neurons, neurons that may play a role in specific circuitry and behaviors.

Keywords: calbindin, calretinin, parvalbumin, VGluT2, VTA

Graphical Abstract

graphic file with name nihms-1036454-f0001.jpg

Ventral tegmental area glutamatergic (VGluT2) neurons differentially express calcium-binding proteins. A subpopulation of ventral tegmental area VGluT2 neurons expressing calbindin (VGluT2-calbindin) target the nucleus accumbens (nAcc) shell.

INTRODUCTION

The VTA has long been considered as a dopaminergic structure, but several studies have demonstrated that the VTA also has GABAergic neurons expressing vesicular GABA transporter (VGaT) mRNA (Nagai et al., 1983; Kosaka et al., 1987; Olson & Nestler, 2007; Margolis et al., 2012; Root et al., 2018b) and glutamatergic neurons expressing VGluT2 mRNA (Kawano et al., 2006; Yamaguchi et al., 2007; Yamaguchi et al., 2011; Morales & Margolis, 2017). Dopaminergic neurons, as defined by the expression of tyrosine hydroxylase (TH), are interspersed with VGluT2 neurons throughout all the VTA nuclei (parabrachial pigmented nucleus, PBP; paranigral nucleus, PN; paraintrafascicular nucleus, PIF; rostral linear nucleus, RLi; interfascicular nucleus, IF and caudal linear nucleus, CLi), but VGluT2 neurons are more prevalent within the midline nuclei and outnumber dopaminergic neurons in the rostral and medial levels of the VTA (Yamaguchi et al., 2007; Yamaguchi et al., 2011). Recent evidence demonstrates that in addition to dopaminergic, GABAergic and glutamatergic neurons, the VTA contains combinatorial neurons that co-release glutamate and dopamine (Zhang et al., 2015) or glutamate and GABA (Root et al., 2014b; Root et al., 2018b).

At the anatomical level, several neuronal markers have been used to further characterize VTA dopaminergic and GABAergic neurons, including detection of the three known calcium-binding proteins (calbindin, CB; calretinin, CR or parvalbumin, PV). It has been estimated that about half of the population of VTA TH-neurons coexpresses CB (Rogers, 1992; Liang et al., 1996) or CR (Rogers, 1992; Liang et al., 1996), and findings from single cell analysis have shown that CB mRNA levels are more abundant in TH neurons from VTA than those from substantia nigra compacta (Chung et al., 2005; Greene et al., 2005). Moreover, retrograde tract tracing studies have shown that VTA TH neurons that coexpress CB (TH-CB neurons) innervate the nucleus accumbens (nAcc, Tan et al., 1999; Barrot et al., 2000). In contrast, it has been reported that VTA GABAergic neurons rarely coexpress CB or CR, but frequently coexpress PV (Olson & Nestler, 2007). Regarding VTA VGluT2 neurons, it is currently unknown the extent to which these neurons coexpress any of the known calcium-binding proteins.

In the present study, we determined whether VTA VGluT2 neurons coexpress CB, CR or PV. By combining radioactive in situ hybridization (to detect VGluT2 mRNA) and immunohistochemistry (to detect CB, CR or PV), we observed that VTA VGluT2 neurons coexpressed CB, CR or PV. We found that about one-third of the population of VTA VGluT2 neurons coexpressed CB, but VTA VGluT2 neurons infrequently coexpressed PV or CR. We determined that within the total population VTA CB neurons, one-third of the VTA CB neurons coexpressed VGluT2 and about half of population of VTA PV neurons coexpressed VGluT2. Given that some VTA VGluT2 neurons coexpress TH or VGaT, we examined whether these neurons coexpressed CB and found that a subpopulation of VTA VGluT2-CB neurons coexpressed either TH or VGaT. Using tract tracing, we further established that some VGluT2-CB neurons project to nAcc.

MATERIAL AND METHODS

Tissue preparation for anatomical studies.

Ten adult male Sprague-Dawley rats (300–350 g body weight, from the Charles River Laboratories) were anesthetized with chloral hydrate (35 mg/100 g) and transcardially perfused with 4% (W/V) paraformaldehyde (PF) in 0.1 M phosphate buffer (PB), pH 7.3. Brains were left in 4% PF for 2 h at 4°C, rinsed with PB and transferred sequentially to 12%, 14% and 18% sucrose solutions in PB. Coronal serial sections of 12 μm in thickness were prepared. All animal procedures were approved by the NIDA Animal Care and Use Committee and experiments were carried out in accordance with the Guidelines laid down by the NIH regarding the care and use of animals for experimental procedures.

Combination of in situ hybridization and immunolabeling.

Coronal free-floating sections (12 µm thickness) from bregma −4.92 mm to −6.48 mm were processed as described previously (Morales & Wang, 2002; Wang & Morales, 2008). Briefly, sections were incubated for 10 min in PB containing 0.5% Triton X-100, rinsed 3 × 10 min with PB, treated with 0.2 N HCl for 15 min, rinsed 3 × 10 min with PB and then acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0 for 10 min. Sections were rinsed 3 × 10 min with PB, post-fixed with 4% paraformaldehyde for 10 min. Prior to hybridization and after a final rinse with PB, the free-floating sections were incubated in hybridization buffer (50% formamide; 10% dextran sulfate; 5x Denhardt’s solution; 0.62 M NaCl; 50 mM DTT; 10 mM EDTA; 20 mM PIPES, pH 6.8; 0.2% SDS; 250 μg/ml salmon sperm DNA; 250 μg/ml tRNA) for 2 h at 55°C. Sections were hybridized for 16 h at 55°C in hybridization buffer containing [35S]- and [33P]-labeled single-stranded antisense or sense of rat VGluT2 (nucleotides 317–2357, Accession # NM-053427). Sections were treated with 4 μg/ml RNase A at 37°C for 1h, washed with 0.5 x SSC, 50% formamide at 55°C for 2h, and with 0.1 x SSC at 60°C for 1 h. After the last SSC wash, sections were rinsed with PB buffer and incubated for 1 h in PB supplemented with 4% bovine serum albumin and 0.3% Triton X-100. This was followed by the overnight incubation at 4°C with one of the primary antibodies, an anti-TH mouse monoclonal antibody (1:1000, MAB 318, Millipore, Billerica, MA) for which specificity has been documented (Tagliaferro and Morales, 2008), anti-calbindin (1:500, CB38, Swant, Switzerland), anti-calretinin (1:1000, 7699/3H, Swant, Switzerland), or anti-parvalbumin (1:500, PVG214, Swant, Switzerland). After rinsing 3 × 10 min in PB, sections were incubated in the biotinylated secondary antibody (1:200) for 1 h at room temperature (RT). Following secondary antibody incubation, sections were rinsed with PB, 3 times for 10 minutes each and incubated in avidin-biotinylated complex (ABC, Vector Laboratories, Burlingame, CA) for 1h at RT. Sections were rinsed, and the peroxidase reaction was then developed with 0.05% 3, 3-diaminobenzidine-4 HCl (DAB) and 0.03% hydrogen peroxide (H2O2). Free-floating sections were mounted on coated slides. Slides were dipped in Ilford K.5 nuclear tract emulsion (Polysciences, Inc., Warrington; 1:1 dilution in double distilled water) and exposed in the dark at 4°C for four weeks prior to development.

Combination of RNAscope and immunolabeling.

To determine whether VGluT2-CB neurons coexpress TH-IR or VGaT mRNA, we obtained coronal free-floating sections (16 µm thickness) from bregma −4.92 mm to −6.48 mm, which we processed for simultaneous immunodetection of TH and CB and transcripts encoding of VGluT2 and VGaT mRNAs using RNAscope. Sections were processed for immunolabeling as described above. Biotinylated secondary antibodies were replaced with fluorochrome tagged antibodies. CB-antibody complexes were detected with secondary Donkey anti-Goat Alexa Fluor 488 antibodies (1:100, 705–545-147, Jackson ImmunoResearch) and TH-antibody complexes with secondary Donkey anti-Mouse Alexa Fluor 750 antibodies (1:100, 175738, AbCam). Sections were mounted onto Fisher SuperFrost slides and dried overnight at 60ºC. RNAscope was performed according to the manufacturer’s instructions. Briefly, sections were treated with heat and protease digestion followed by hybridization with two fluorescent probes, one for detection of VGluT2 mRNA (Atto 550) and other for detection of VGaT mRNA (Atto 647).

Retrograde neuronal tract tracing.

Male Sprague-Dawley rats (9 weeks, 300–400 grams, Charles River Laboratories, n=5) were used for retrograde tracing. All animal procedures were performed in accordance with NIH guidelines and approved by the NIDA animal care and use committee. The retrograde tract tracer Fluoro-Gold (FG; 1% in cacodylate buffer, pH 7.5, Fluorochrome) was delivered unilaterally into the nAcc shell (n = 5 rats; coordinates relative to bregma in mm at 10o mediolateral angle: AP +1.7, ML −2.5, DV −7.7). FG was delivered iontophoretically through glass micropipette (42–55 µm) by applying 3 µA current in 5 s pulses at 10 s intervals for 18–20 min. The micropipette was left in place for an additional 10 min to prevent backflow. One week following the FG injection, rats were anesthetized and perfused transcardially with fixative solution [4% paraformaldehyde]. Coronal serial sections of 16 µm for VTA and 30 µm for nAcc shell were prepared. Serial sections from nAcc shell (n = 5 rats) were processed to determine the injection spread.

Phenotyping of retrogradely labeled neurons.

nAcc shell (n = 3 rats) with confined FG injections were processed for RNAscope and immunohistochemistry for further analysis. To determine if FG neurons in the VTA coexpress VGluT2 mRNA and CB-IR, coronal free floating sections (16 µm thickness) from −4.92 mm to −6.48 mm were processed for simultaneous detection of FG, TH and CB-IR using immunolabeling and transcript encoding of VGluT2 mRNA using RNAscope. Sections were processed for simultaneous immunolabeling and RNAscope as described above. FG was detected by rabbit anti-FG (1:100, AB153–1, Millipore) and Donkey anti-Rabbit Alexa Fluor 750 (1:100, 175728, Abcam). TH was detected by Donkey anti-Mouse 647 (1:100, 715–605-151, Jackson ImmunoResearch).

Data Analysis of Cellular Subpopulations.

Radioactive in situ hybridization sections were viewed, analyzed, and photographed with bright field or epiluminescence microscopy using an Olympus BX51microscope fitted with 4X and 20X objective lenses. Single and double-labeled neurons were observed within each traced region at high power (20X Objective lens) and marked electronically. Subdivisions of the midbrain dopamine system were traced as previously described (Sanchez-Catalan et al., 2014; Morales & Margolis, 2017). Double-labeled sections (VGluT2-CB, VGluT2-CR and VGluT-PV) were analyzed using epiluminescence to increase the contrast of silver grains (neither dark-field nor bright field optics allow clear visualization of silver grains when colocalized with high concentration of immunoproducts). A cell was considered to express transcripts encoding VGluT2 when its soma contained concentric aggregates of silver particles above background level. A neuron was considered to express TH, CB, CR, or PV immunoreactivity when its soma was clearly labeled as brown. An immunolabeled neuron was included in the calculation of total population of immunolabeling cells when the stained cell was at least 5 μm in diameter. The cells expressing VGluT2 mRNA, CB−, CR−, or PV− immunoreactivity, or both markers were counted separately. To determine cellular coexistence of VGluT2 mRNA and CB−, CR−, PV− immunoreactivity (IR), (a) silver grains corresponding to VGluT2 expression were focused under epiluminescence microscopy, (b) the path of epiluminescence light was blocked without changing the focus, (c) bright field light was used to determine if a brown neuron, expressing CB, CR or PV in focus, contained the aggregates of silver grains seen under epiluminescence. The background was evaluated from slides hybridized with sense probes. RNAscope sections were viewed, analyzed, and photographed with an Olympus FV1000 confocal microscope. Negative control hybridizations showed negligible fluorophore expression. Labeled cells were counted 3 times, each time by a different observer. Pictures were adjusted to match contrast and brightness by using the program Adobe Photoshop CS7 (Adobe Systems Incorporated, Seattle, WA).

Statistical Analysis.

Statistical analyses were performed using GraphPad Prism version 5.04 software for Windows (GraphPad Software, La Jolla, California, USA). We used a one-way repeated measures analysis of variance (ANOVA), followed by post-hoc Newman-Keuls multiple comparison test; a level of 0.05 was required to reach statistical significance. We calculated the frequency and distribution of dual VGluT2-CB, VGluT2-CR and VGluT2-PV neurons (a) in each VTA nuclei: RLi, IF, PN, PIF, PBP and CLi and, (b) at different rostro-caudal levels: rostral (−4.92 to −5.04 mm), medial (−5.04 to −5.45 mm) and caudal (−5.45 to −6.48 mm) in the VTA.

Accordingly, we used statistical analysis to determine significant differences in frequency and distribution of dual VGluT2-CB, VGluT2-CR and VGluT2-PV neurons between (a) VTA nuclei and, (b) bregma levels.

RESULTS

By radioactive in situ hybridization, we replicated our earlier findings and confirmed the presence of VGluT2 expressing neurons within each nucleus of the VTA with higher concentration in the midline nuclei and lower concentration in lateral nuclei (Fig. 1). We detected high concentration of neurons expressing tyrosine hydroxylase immunoreactivity (TH-IR; Fig. 1B and 2B), calbindin immunoreactivity (CB-IR; Fig. 1B’) or calretinin immunoreactivity (CR-IR; Fig. 2B’) throughout the medial and lateral nuclei of the VTA. In contrast, we found that neurons expressing parvalbumin immunoreactivity (PV-IR) were concentrated in the midline nuclei of the VTA (Fig. 3B).

Figure 1. Cellular coexpression of VGluT2 mRNA and Calbindin immunoreactivity (CB-IR) in the rat VTA (−5.28 mm from bregma).

Figure 1

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Coronal brain sections were processed for the simultaneous detection of CB by immunolabeling and VGluT2 mRNA by in situ hybridization. (A), Section at low magnification showing CB-IR (dark brown label) under bright-field microscopy. (B-B’’), Delimited area in A is shown at higher magnification. (B), TH immunoreactivity (TH-IR) highlighting the VTA boundaries; (B’), CB-IR; and (B’’), VGluT2 mRNA signal, seen as aggregates of silver grains. Cellular signal for CB-IR and VGluT2 mRNA are seen in the parabrachial pigmented nucleus (PBP), paranigral nucleus (PN) the rostral linear nucleus of the raphe (RLi) and interfascicular nucleus (IF). (C-C”), CB-IR neurons coexpressing VGluT2 mRNA (arrows) in the RLi nucleus (pink box). (D-D’’), CB-IR neurons lacking VGluT2 mRNA (arrowheads) in the PBP nucleus (blue box). The CB-IR neurons are seen as brown and the VGluT2 mRNA signal as black grain aggregates under bright field microscopy and as silver grain aggregates under epiluminescence microscopy. fr, fasciculus retroflexus; mp, mammillary peduncle; mt, medial terminal nucleus of the accessory optic tract; mtg, mamillotegmental tract; Scale bar shown in D” is 750 µm for A; 100 µm for B-B’’; 12.5 µm for C-D”.

Figure 2. Cellular expression of VGluT2 mRNA and Calretinin immunoreactivity (CR-IR) in the rat VTA (−5.28 mm from bregma).

Figure 2

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Coronal brain sections were processed for the simultaneous detection of CR by immunolabeling and VGluT2 mRNA by in situ hybridization. (A), Section at low magnification showing CR-IR (dark brown label) and VGluT2 mRNA signal seen as aggregates of black grains in the PBP, PN, RLi and IF nuclei. (B-B’’), Delimited area in A is shown at higher magnification. (B), TH immunolabeling highlighting the boundaries of the VTA; (B’), CR-IR and VGluT2 mRNA; (B’’), VGluT2 mRNA signal seen as aggregates of silver grains. (C and C’), CR-IR neurons lacking VGluT2 mRNA (arrowheads) and VGluT2 mRNA neurons lacking CR-IR (arrows) in the RLi nucleus (blue box). Scale bar shown in C’ is 750 µm for A; 100 µm for B-B’’; 12.5 µm for C-C’.

Figure 3. Cellular coexpression of VGluT2 mRNA and Parvalbumin immunoreactivity (PV-IR) in the rat VTA.

Figure 3

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Coronal brain sections were processed for the simultaneous detection of PV by immunolabeling and VGluT2 mRNA by in situ hybridization seen in the PBP, PN, RLi and IF nuclei. (A), Section at low magnification showing PV-IR (top) and TH immunoreactivity (TH-IR, bottom) (dark brown label) and VGluT2 mRNA signal (top) seen as aggregates of green grains. (B – B’’), Delimited area in A is shown at higher magnification. (B), PV-IR and (B’), VGluT2 mRNA signal seen as aggregates of silver grains. (C-C’’), PV-IR neuron coexpressing VGluT2 mRNA (arrows) in the PN nucleus (pink box). (D-D’’), PV-IR neurons lacking VGluT2 mRNA (arrowheads) in the PBP nucleus (blue box). (E – G’), Coronal brain sections were processed for simultaneous detection of PV-IR and PV mRNA. (E), PV-IR (dark brown label) and PV mRNA signal is seen as black grains. (F-F’), Delimited area in E is shown at higher magnification. (F), PV-IR and (F’), PV mRNA signal seen as silver grains. (G – G’’), PV-IR neuron coexpressing PV mRNA (arrows) in the PN. Note that all PV-IR neurons coexpress PV mRNA, indicating the specificity of the PV antibody. Scale bar shown in A is 1000 µm and scale bar shown in G’’ is 750 µm for E; 100 µm for B, B’, F, F’; 12.5 µm for C-D” and G-G’’.

One third of the total population of VTA VGluT2 neurons coexpressed CB-IR.

We detected expression of CB-IR in VGluT2 neurons within each nucleus of the VTA (RLi, IF, PIF, CLi, PN and PBP; Fig. 1B’). We next determined the frequency of VTA neurons coexpressing VGluT2 mRNA and CB by analyzing an average of 23 ± 2 sections per rat (12 µm in thickness; 4 rats), sections that included the different nuclei of the VTA. By analysis of coexpression of VGluT2 mRNA and CB-IR, we found that 29.6 ± 0.9% of the VGluT2 mRNA expressing neurons coexpressed CB-IR (3, 201 VGluT2-CB neurons from a total of 10, 990 VGluT2 neurons; Fig. 4). These dual VGluT2-CB neurons represented 33.8 ± 1.55% of the total population of counted VTA CB-IR neurons (9, 354 CB-IR neurons; Fig. 5A).

Figure 4. Frequency of VTA neurons expressing VGluT2 mRNA and those that coexpress CB, CR or PV.

Figure 4

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(A), Within the total population of the VGluT2 mRNA expressing neurons, on average, 30% coexpressed CB, 0.9% coexpressed CR and 3% coexpressed PV. The total number of neurons counted is indicated in parentheses. (B-C), VTA rostro-caudal gradient of distribution of neurons expressing CB, PV and those coexpressing VGluT2 and CB or VGluT2 and PV. Scattered plots represent the total number (mean ± S.E.M.) of counted neurons from four rats. (B), Total number of CB-IR neurons and VGluT2-CB neurons (neurons coexpressing VGluT2 mRNA and CB-IR) was highest in the medial aspects of the VTA. (C), Total number of PV-IR neurons and VGluT2-PV neurons (neurons coexpressing VGluT2 mRNA and PV-IR) was highest in the medial VTA.

Figure 5. Frequency of VTA neurons expressing CB, CR or PV and those that coexpress VGluT2 mRNA and distribution of VGluT2-CB, VGluT2-CR and VGluT2-PV neurons within the different nuclei of the VTA.

Figure 5

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(A), Within the total population of CB-IR neurons, almost one-third coexpressed VGluT2 mRNA. 33% of VGluT2-CB neurons were detected in the IF nucleus. (B), CR-IR neurons rarely coexpressed VGluT2 mRNA in the VTA. 37% of VGluT2-CR neurons were detected the in the RLi nucleus (C), Within the total population of PV-IR neurons, almost half coexpressed VGluT2 mRNA. 58% of VGluT2-PV neurons were detected in the PN nucleus. [Average percentage was determined from four rats and scattered plots represent the total number (mean ± S.E.M.)]. (D – G), Frequency of VGluT2-CB and VGluT2-PV neurons at bregma level −5.28 mm. (D – E), On average, more than 80% of the total population of CB-IR neurons coexpressed VGluT2 mRNA in the IF and RLi nuclei. (F – G), Almost 56% of neurons in the IF nucleus and more than half of PV-IR neurons in the RLi nucleus coexpressed VGluT2 mRNA. (Average percentage was determined from three rats).

By analyzing the number and frequency of dual VGluT2-CB neurons within each nucleus of the VTA, we detected the highest frequency of VGluT2-CB neurons in the IF, medial PBP, RLi and PN nuclei, with a frequency of 32.7 ± 3.46% (n = 1068 neurons, counted from 4 rats) in the IF nucleus; 21.68 ± 2.4% (n = 708 neurons) in the medial PBP nucleus; 20.83 ± 2.87% (n = 639 neurons, counted from 4 rats) in the RLi nucleus and 14.5 ± 1.7% (n = 501 neurons, counted from 4 rats) in the PN nucleus. We found the lowest frequency of dual VGluT2-CB neurons in the PIF and CLi nuclei, 7.82 ± 1.6% (n = 217 neurons, counted from 4 rats) in the PIF nucleus and 2.37 ± 0.8% (n = 68 neurons, counted from 4 rats) in the CLi nucleus (Fig. 5A). By one-way ANOVA analysis, we found statistical differences in frequency and distribution of VGluT2-CB neurons between different VTA nuclei (F(5,23) = 7.53; p<0.001). By post hoc Newman-Keuls analysis we found that dual VGluT2-CB neurons were significantly more concentrated in the IF nucleus than in the PN (p<0.05), PIF (p<0.01), or CLi (p<0.001) nuclei (Fig. 5A).

We next analyzed the VTA rostro-caudal distribution and frequency of dual VGluT2-CB neurons within the total population of VTA CB-IR neurons. We detected the highest concentration of dual VGluT2-CB neurons in the IF and RLi nuclei at the bregma level of −5.28 mm (Fig. 5D and E). At this bregma level within the IF nucleus, we found that 82 ± 7.8% of the CB-IR neurons coexpressed VGluT2 mRNA (169 dual VGluT2-CB neurons from a total of 201 CB-IR neurons, counted from 3 rats), which corresponded to 57.62 ± 6.7% of the total number of detected VGluT2 neurons (n = 283 neurons, counted from 3 rats) within the IF nucleus (Fig. 5D). At the same bregma level within the RLi nucleus, we found that 89.1 ± 2.3% of the CB-IR neurons coexpressed VGluT2 mRNA (125 VGluT2-CB neurons from a total of 140 CB-IR neurons, counted from 3 rats), which correponded to 32.58 ± 3.35 of the total number of detected VGluT2 neurons (n = 379 neurons, counted from 3 rats) within the RLi nucleus (Fig. 5E).

As a follow up, we examined and compared the VTA rostro-caudal distribution and frequency of dual VGluT2-CB neurons within the VTA. We observed higher number of dual VGluT2-CB neurons (46.2 ± 7.29%, 1583 neurons, counted from 4 rats) in the medial aspects of the VTA (bregma; −5.04 to −5.45 mm), followed by caudal (bregma; −5.45 to −6.48 mm; 44.54 ± 6.95%, 1382 neurons, counted from 4 rats) and then rostral aspects (bregma; −4.92 to −5.04 mm; 9.24 ± 2.67%, 236 neurons, counted from 4 rats; Fig. 4B). By one-way ANOVA analysis, we did not find statistical differences in frequency and distribution of dual VGluT2-CB neurons between three bregma levels (F(2,11) = 4.32; p = 0.07).

In summary, we found that throughout the rostro-caudal levels of the VTA, one-third of the VTA VGluT2 neurons coexpressed CB, these dual VGluT2-CB neurons were concentrated in the IF nucleus, followed by the medial PBP, RLi, PN nuclei and least frequently in the CLi nucleus within the VTA (Fig. 5A and 6). Moreover, we determined that the vast majority of CB-IR neurons located in the medial nucleus of the VTA coexpressed VGluT2 mRNA.

Figure 6. Summary diagram of the distribution of the VGluT2 neurons coexpressing CB-IR, CR-IR or PV-IR within each subdivision of the rat VTA.

Figure 6

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(A – D), Distribution of the VGluT2-CB neurons (neurons coexpressing VGluT2 mRNA and CB-IR). These neurons are present throughout the rostro-caudal levels of each subdivision of the VTA with a medio-lateral decreasing gradient of concentration. (A’ – D’), Distribution of the VGluT2-CR neurons (neurons coexpressing VGluT2 mRNA and CR-IR). (A’’ – D’’), Distribution of VGluT2-PV neurons (neurons coexpressing VGluT2 mRNA and PV-IR). Each panel represents the average number of labeled neurons found in four sections, each section from a different rat. fr, fasciculus retroflexus; IF, interfascicular nucleus; mp, mammillary peduncle; mt, medial terminal nucleus of the accessory optic tract; mtg, mamillotegmental tract; PBP, parabrachial pigmented nucleus; PN, paranigral nucleus; RLi, rostral linear nucleus of the raphe; CLi, caudal linear nucleus of the raphe; RRF, retrorubral field; VTAR, ventral tegmental area, rostral part.

VTA VGluT2 neurons rarely coexpressed CR-IR or PV-IR.

We detected neurons expressing VGluT2 mRNA intermingled with CR-IR neurons throughout the rostro-caudal aspects of the VTA (Fig. 2), but VGluT2 mRNA expressing neurons rarely coexpressed CR (Fig. 2B’). We calculated a frequency of 0.9 ± 0.16% dual VGluT2-CR neurons (57 neurons, counted from 4 rats) within the total population of counted VTA VGluT2 neurons (6, 940 VGluT2 neurons, counted from 4 rats; Fig. 4). We further determined that these dual VTA VGluT2-CR neurons represented 0.4 ± 0.07% of the total population of counted VTA CR-IR neurons (14, 979 CR-IR neurons; Fig. 5B). The infrequent number of dual VGluT2-CR neurons (20 neurons, counted from 4 rats) were concentrated in the RLi nucleus (Fig. 5B), corresponding to 37.5 ± 5.06% of the total number of VTA VGluT2-CR neurons (57 neurons, counted from 4 rats). By one-way ANOVA analysis, we found statistical differences in frequency and distribution of VGluT2-CR neurons between different VTA nuclei (F(5,23) = 7.98; p<0.001). By post hoc Newman-Keuls analysis, we found that dual VGluT2-CR neurons were significantly more concentrated in the RLi nucleus than in the PN (p<0.05) or PIF (p<0.01) nuclei (Fig. 5B).

Next, we examined and compared the VTA rostro-caudal distribution and frequency of dual VGluT2-CR neurons within the VTA and although, we observed dual VGluT2-CR neurons concentrated in the medial aspects of the VTA (bregma; −5.04 to −5.45 mm; 65.25 ± 10.15%, 39 neurons), followed by rostral (bregma; −4.92 to −5.04 mm; 22.60 ± 13.72%, 19 neurons) and then caudal aspects (bregma; −5.45 to −6.48 mm; 12.16 ± 4.61%, 7 neurons). By one-way ANOVA alaysis, we found statistical differences in frequency and distribution of dual VGluT2-CR neurons between three bregma levels (F(2,11) = 5.14; p = 0.05). By post hoc Newman-Keuls analysis, we found that dual VGluT2-CR neurons were significantly more concentrated in the medial aspects compared to the caudal (p<0.05) of the VTA.

We also detected neurons expressing VGluT2 mRNA intermingled with PV-IR neurons throughout the rostro-caudal aspects of the VTA, concentrated in the medial nuclei (Fig. 3). By analysis of coexpression of VGluT2 mRNA and PV-IR, we found that 2.5 ± 0.2% of the VGluT2 neurons coexpressed PV-IR (354 VGluT2-PV neurons from a total of 14, 294 VGluT2 neurons, counted from 4 rats; Fig. 3C and 4A). While we determined that the dual VTA VGluT2-PV neurons were a fraction of the total population of VTA VGluT2 mRNA expressing neurons, they corresponded to 51.95 ± 7.75% of the total population of counted VTA PV-IR neurons (697 PV-IR neurons; Fig. 5C). Within the total population of dual VGluT2-PV neurons (354 neurons), we found that 58.2 ± 3.4% (n = 209 neurons) were present in the PN nucleus, 18.1 ± 4.68% (n= 61 neurons) in the PBP nucleus, 14.6 ± 1.74% (n= 53 neurons) in the IF nucleus, 4.85 ± 1.09% (n= 17 neurons) in the RLi nucleus and 3.8 ± 0.9% (n = 14 neurons) in the PIF nucleus (Fig. 5C). We did not detect VGluT2-PV neurons in the CLi nucleus. By one way ANOVA analysis, we found statistical differences in frequency and distribution of VGluT2-PV neurons between different VTA nuclei (F(5,23) = 60.95; p<0.001). By post hoc Newman-Keuls analysis, we found that dual VGluT2-PV neurons were significantly more concentrated in the PN nucleus than in the IF, RLi, PBP or PIF (p<0.001) nuclei. VGluT2-PV neurons were also significantly higher in number in the IF nucleus compared to the RLi nucleus (Newman-Keuls test, p<0.05; Fig. 5C).

We then analyzed the VTA rostro-caudal distribution and frequency of dual VGluT2-PV neurons within the total population of VTA PV-IR neurons. We detected dual VGluT2-PV neurons in the IF and RLi nuclei at the bregma level of −5.28 mm (Fig. 5F and G). At this bregma, we detected an average of 55.56 ± 29.4% PV-IR neurons coexpressing VGluT2 mRNA (4 VGluT2-PV neurons out of 8 PV-IR neurons, neurons counted from 3 rats), which corresponded to 2.01 ± 1.21% of total number of counted VTA VGluT2 neurons (n = 276 neurons, from 3 rats) within the IF nucleus (Fig. 5F), and 52.8 ± 12.11% of the PV-IR neurons that coexpressed VGluT2 mRNA (5 VGluT2-PV neurons from a total of 9 PV-IR neurons), corresponding to 1.39 ± 0.44% of counted VGluT2 neurons within the RLi nucleus (n = 385 neurons, counted from 3 rats; Fig. 5G). At this bregma, we also analyzed the frequency of dual VGluT2-PV neurons, and detected that 86.9 ± 7.24% of the PV-IR neurons coexpressed VGluT2 mRNA (15 VGluT2-PV neurons from a total of 17 PV-IR neurons), corresponding to 2.67 ± 0.46% of the counted VGluT2 neurons (n = 625 neurons, from 3 rats) within the PN nucleus.

As a follow up, we analyzed and compared the VTA rostro-caudal distribution and frequency of dual VGluT2-PV neurons within the VTA and observed highest number of VGluT2-PV neurons in the medial aspects of the VTA (bregma; −5.04 to −5.45 mm; 60.91 ± 2.6%, 215 neurons, counted from 4 rats), followed by caudal (bregma; −5.45 to −6.48 mm; 34.11 ± 2.62%, 122 neurons, counted from 4 rats) and then rostral aspects (bregma; −4.92 to −5.04 mm; 4.99 ± 2.29%, 17 neurons, counted from 4 rats). By one-way ANOVA analysis, we found statistical differences in frequency and distribution of VGluT2-PV neurons between different bregma levels, (F(2,11) = 51.44; p<0.001). By post hoc Newman-Keuls analysis, we found that dual VGluT2-PV neurons were significantly more concentrated in the medial aspects compared to the caudal (p<0.01) and rostral aspects (p<0.001) of the VTA. VGluT2-PV neurons were also significantly higher in number in the caudal aspects than the rostral aspects of the VTA (p<0.01; Fig. 4C). Thus, we found that a small fraction of VTA VGluT2 neurons coexpressed PV. Notably, these dual VGluT2-PV neurons represented more than half of the PV-IR neurons detected in the VTA. Within the entire rostro-caudal aspect of the VTA, these dual VGluT2-PV neurons were concentrated in the PN nucleus followed by PBP nucleus and then IF, RLi and PIF nuclei (Fig. 5C).

In summary, we detected within the VTA a latero-medial increasing gradient of distribution of VGluT2 neurons coexpressing CB, CR or PV (Fig. 6). These VGluT2-CB, VGluT2-CR and VGluT2-PV neurons were concentrated in the RLi and IF nuclei in the anterior aspects of the VTA (Fig. 5 and 6), distributed in the RLi, IF, PBP and PN nuclei in the medial aspects of the VTA, and in the CLi and IF nuclei in the caudal aspects of the VTA.

A subset of VTA VGluT2-CB neurons coexpressing TH-IR innervate the nucleus accumbens shell.

We have previously demonstrated that some VTA rat VGluT2 neurons coexpress TH (VGluT2-TH neurons; Yamaguchi et al., 2011) and others coexpress VGaT mRNA (VGluT2-VGaT neurons; Root et al., 2014b). Thus we next determined whether VTA VGluT2-CB are part of the subpopulation of VGluT2-TH or VGluT2-VGaT neurons by using a combination of immunolabeling (to detect TH and CB; Fig. 7A and 7B) and RNAscope (to detect VGluT2 mRNA and VGaT mRNA; Fig. 7C and 7D). We found that within the total population of VGluT2-CB neurons (506 neurons, counted from 4 rats) about 20% coexpressed TH-IR (18.56 ± 3.66%, n = 88 neurons; Fig. 7H and 7K), more than 10% coexpressed VGaT mRNA (12.61 ± 1.24%, n = 62 neurons; Fig. 7I’ and 7K), and 6.2 ± 1.64% coexpressed TH-IR and VGaT mRNA (Fig. 7K).

Figure 7. Detection of tyrosine hydroxylase immunoreactivity (TH-IR) or VGaT mRNA in VGluT2-CB neurons in the rat VTA.

Figure 7

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Coronal brain sections were processed for the simultaneous detection of TH (cyan) and CB (green) by immunolabeling and VGluT2 (red) and VGaT (white) mRNAs by RNAscope (low magnification, A-E). Delimited areas are shown at higher magnification. (F – J), VGluT2-CB neuron coexpressing TH-IR (white arrow). (F’ – J’), VGluT2-CB neurons coexpressing VGaT mRNA (pink arrows). K, Average percentage of total VGluT2-CB neurons lacking or coexpressing TH or VGaT. The total number of neurons counted is indicated in parentheses (counted from 4 rats). Scale bar shown in E is 150 µm for A-E and scale bar shown in J’ is 10 µm for F-J’.

We and others have previously demonstrated that VTA VGluT2 neurons innervate the nucleus accumbens (nAcc, Yamaguchi et al., 2011; Taylor et al., 2014) and that some of these VGluT2 neurons innervating the nAcc coexpress TH (VGluT2-TH; Stuber et al., 2010; Tecuapetla et al., 2010; Yamaguchi et al., 2011; Zhang et al., 2015), preferentially targeting the shell of the nAcc (Poulin et al., 2018). In here we showed that a subset of VTA VGluT2-CB neurons coexpress TH (VGluT2-CB-TH; Fig. 7), so we first determined whether VTA VGluT2-CB neurons innervate nAcc shell (Fig. 8) by locally injecting Fluoro-Gold (FG, Fig. 8A and 8B), and then phenotyped these VTA FG neurons. We detected 1, 268 VTA FG neurons (counted from 3 rats; Fig. 8C) from which about one-third coexpressed VGluT2 mRNA (FG-VGluT2, n = 376 neurons, counted from 3 rats; Fig. 8D and 8G). From this total population of FG-VGluT2 neurons (n = 376 neurons), we found that the majority lacked CB-IR (73.6 ± 4.14%, n = 286 neurons, counted from 3 rats), and about one-quarter coexpressed CB-IR (FG-VGluT2-CB, 26.4 ± 4.1%, n = 90 neurons, counted from 3 rats; Fig. 8L). Given that some VTA VGluT2-CB neurons coexpress TH (VGluT2-CB-TH neurons), we next determined whether VGluT2-CB-TH neurons innervate the nAcc shell and we found that within the total population of FG-VGluT2-CB neurons (n = 90 neurons, counted from 3 rats), 83.3 ± 14% coexpressed TH-IR (FG-VGluT2-CB-TH, n = 64 neurons; Fig. 8J and 8L) and 16.7 ± 14% lacked TH-IR (n = 26 neurons; Fig. 8J’ and 8L), indicating that the majority of VGluT2-CB neurons coexpressing TH innervate the nAcc shell.

Figure 8. Some VTA VGluT2-CB neurons target nAcc shell.

Figure 8

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(A), Retrograde tracer FG was delivered into the nAcc shell. (B), FG injection site in the nAcc shell. (C – F), Low magnification of VTA coronal section processed for the simultaneous detection of FG (white), TH (cyan) and CB (green) by immunolabeling and VGluT2 (red) mRNA by RNAscope. Delimited areas are shown at higher magnification. (G – K), FG-IR neuron coexpressing VGluT2 mRNA, CB-IR and TH-IR (pink arrow). FG-IR neuron coexpressing CB-IR and TH-IR (yellow arrow). (G’ – K’), FG-IR neuron coexpressing VGluT2 mRNA and CB-IR (white arrow). (L), Average percentage of FG-VGluT2 neurons lacking or coexpressing CB and proportion of FG-VGluT2-CB neurons coexpressing or lacking TH. The total number of neurons counted is indicated in parentheses (counted from 3 rats). Roughly one-quarter of FG-VGluT2 neurons show coexpression of CB and majority of these neurons coexpress TH. Scale bar shown in B is 250 µm; scale bar shown in F is 150 µm for C-F; and scale bar shown in K’ is 10 µm for G-K’.

In summary, our findings show that calbindin is the main calcium-binding protein present in VTA VGluT2 neurons and a subset of VTA VGluT2 neurons coexpressing calbindin with the capability to co-release glutamate and dopamine innervate the nucleus accumbens shell.

DISCUSSION

In here, we demonstrated coexpression of CB, CR or PV within VGluT2-neurons located in the midline nuclei of the rat VTA. We found that one third of the total population of VTA VGluT2-neurons coexpresses CB, but VTA VGluT2 neurons rarely coexpress CR or PV. Although the total number of PV neurons is low in the VTA, half of the total population of these PV neurons coexpresses VGluT2. These findings further provide evidence for molecular diversity among VTA VGluT2-neurons, a population of neurons that may participate in important brain pathways and specific aspects of behavior.

We have previously demonstrated that the VTA VGluT2-neurons are diverse at the molecular (Yamaguchi et al., 2007; Yamaguchi et al., 2011; Li et al., 2013; Root et al., 2014b; Root et al., 2018b) and functional levels (Dobi et al., 2010; Root et al., 2014a; Wang et al., 2015; Qi et al., 2016). At the molecular level, some VTA VGluT2-neurons coexpress dopaminergic or GABAergic markers (Yamaguchi et al., 2007; Yamaguchi et al., 2011; Root et al., 2014b), but most VTA VGluT2-neurons lacks these markers (Yamaguchi et al., 2007; Yamaguchi et al., 2011; Root et al., 2014b). The VGluT2-neurons that coexpress TH (VGluT2-TH neurons) are not homogeneous, as some of them coexpress dopamine transporters, dopamine D2 receptors or vesicular monoamine transporters, and others lack these dopamine markers (Li et al., 2013). The dual VGluT2-TH neurons co-release glutamate (Stuber et al., 2010; Tecuapetla et al., 2010) and dopamine (Zhang et al., 2015) within the nucleus accumbens. Moreover, a subpopulation of VTA VGluT2-neurons expresses GABAergic markers, and co-releases glutamate and GABA within the lateral habenula (Root et al., 2014b) from the same axon terminal, but from different pool of vesicles (Root et al., 2018b). At the functional level, some VTA VGluT2-neurons participate in reward (Wang et al., 2015; Yoo et al., 2016), and others participate in aversion (Root et al., 2014a; Qi et al., 2016; Root et al., 2018a). It remains to be determined the extent to which these previously identified molecular and functional diverse VTA VGluT2-neurons overlap with the VTA VGluT2-neurons that we found in the present study to lack or coexpress CB, CR or PV.

VTA neurons expressing CB.

Anatomical findings have shown that within the VTA, CB is expressed mostly in TH neurons (Liang et al., 1996; Nemoto et al., 1999), and infrequently express in GABA neurons (Olson & Nestler, 2007). Here, we demonstrated that one-third of the total population of VTA-CB neurons coexpresses VGluT2 mRNA, indicating that VGluT2-CB neurons are a major subpopulation of neurons within the entire population of VTA-CB neurons. Furthermore, we found that among the total population of VTA-VGluT2 neurons, one third of them expresses CB (VGluT2-CB neurons), showing that CB can be useful as an additional cellular marker for identification of subpopulations of VTA glutamatergic neurons. Moreover, we found that the VTA VGluT2-CB neurons can be further distinguished based on their potential of co-release of neurotransmitter, as we found that about 20% of VGluT2-CB neurons coexpress TH (VGluT2-CB-TH neurons), having the capability to co-release glutamate and dopamine, and about 13% of VGluT2-CB neurons coexpress VGaT (VGluT2-CB-VGaT neurons), having the capability of corelase glutamate and GABA.

While it is unclear the role of CB in cellular function, electrophysiological recordings have demonstrated that VTA dopamine neurons coexpressing CB have a significantly faster pacemaker frequencies with smaller afterhyperpolarizations compared with those lacking CB (Neuhoff et al., 2002). A role for CB in the control of exocytosis in VTA dopamine neurons has been recently suggested (Pan & Ryan, 2012). In these studies, the probability of vesicle exocytosis was increased by CB knockdown in VTA dopamine neurons, but decreased by CB overexpression, suggesting a lower vesicular release probability by VTA dopamine neurons than by those of the substantia nigra (Pan & Ryan, 2012). It remains to be determined whether CB plays a role in controlling the probability of vesicle exocytosis within the different subpopulation of VTA VGluT2-CB neurons identified in the present study (VGluT2-CB, VGluT2-CB-TH, VGluT2-CB-VGaT).

VTA neurons expressing CR or PV.

Previous anatomical studies have demonstrated that within the total population of VTA TH neurons ≈ 64% coexpresses CR (Rogers, 1992), and that ≈ 65% of the total population of VTA CR neurons coexpresses TH (Isaacs & Jacobowitz, 1994). In contrast, CR has not been detected in VTA GABA neurons (Olson & Nestler, 2007). Similarly, we rarely detected expression of CR in VTA VGluT2 neurons. Thus, cellular detection of CR may be useful for the characterizing of subpopulations of VTA dopamine neurons, but not for the characterizing of non-dopamine neurons.

Previous studies have shown the presence of VTA cellular expression of PV, including results from a recent whole brain Drop-seq study (Saunders et al., 2018). A study on cellular characterization of VTA neurons had concluded that ≈ 75% of the PV neurons coexpresses GAD protein (Olson & Nestler, 2007). In this study, GAD protein was detected by immunolabeling after the disruption of cellular trafficking, a process that is known to induce gene upregulation. In contrast, we detected both PV and VGluT2 mRNA under normal conditions, and found that approximately half of the population of VTA PV neurons coexpresses VGluT2 mRNA. Thus the apparent discrepancy between the previous published frequency of VTA PV-GABA neurons and our findings may be explained by methodological differences.

Throughout the brain, anatomical and electrophysiological evidence indicates that PV neurons are mostly inhibitory GABA neurons (Celio & Heizmann, 1981; Celio, 1986; Kawaguchi et al., 1987; Rohrenbeck et al., 1987; Stichel et al., 1988; DeFelipe et al., 1989; Demeulemeester et al., 1989; Hendry et al., 1989; Blumcke et al., 1990; Lewis & Lund, 1990; Van Brederode et al., 1990; Williams et al., 1992). However, several studies have demonstrated expression of VGluT2 in PV neurons in several brain areas (Roccaro-Waldmeyer et al., 2018), including the lateral hypothalamus (Girard et al., 2011; Kisner et al., 2018; Roccaro-Waldmeyer et al., 2018) and the entopeduncular nucleus (Wallace et al., 2017). Moreover, recent behavioral studies have shown that the genetic selective removal of VGluT2 within the total population of VGluT2-PV neurons results in deficits in locomotion and vocalization, decreased thermal nociception, and increase in social dominance (Roccaro-Waldmeyer et al., 2018). Future studies will be necessary to determine whether any of these observed behavioral alterations may be mediated, in part, by the removal of VGluT2 within the population of VTA VGluT2-PV neurons.

VTA VGluT2-CB-TH neurons innervate the nAcc shell.

In common with VTA TH-CB neurons (Tan et al., 1999; Barrot et al., 2000), we found that some VTA VGluT2-CB neurons innervate the nAcc shell. Moreover, we determined that the majority of VTA VGluT2-CB neurons targeting the nAcc shell coexpress TH-IR, as such, having the capability to co-release glutamate and dopamine within the nAcc shell. We have previously demonstrated that VGluT2-TH neurons innervating the nAcc have separate adjacent ultrastructural domains within a single axon for accumulation and release of dopamine and glutamate from independent pools of vesicles (Zhang et al., 2015). Given that calbindin seems to regulate dopamine release from VTA dopamine-calbindin neurons (Pan & Ryan, 2012), this raises the possibility of the participation of calbindin in regulating dopamine release from VTA glutamatergic-CB-dopamine neurons innervating the nAcc shell.

In conclusion, our findings indicate that while most VTA VGluT2-neurons lack expression of calcium-binding proteins, one-third of the VTA VGluT2-neurons coexpresses CB and half of the neuronal population of VTA PV neurons coexpresses VGluT2. These findings provide the basis for future studies towards establishing the neuronal circuitry involving the participation of those VTA VGluT2 neurons that coexpress or lack calcium-binding proteins. Towards this goal, we identified a subpopulation of VTA calbindin neurons with the capability to co-release glutamate and dopamine within the nAcc shell. Our findings broaden our understanding of the molecular diversity among VTA neurons endowed with the capability to use glutamate as neurotransmitter.

ACKNOWLEDGEMENTS

This work was supported by the Intramural Research Program of the National Institute on Drug Abuse. We thank NIDA Visual Media Services for preparation of figure display in the Graphical Abstract.

ABBREVIATIONS

CB

calbindin

CLi

caudal linear nucleus

CR

calretinin

IF

interfascicular nucleus

nAcc

nucleus accumbens

PB

phosphate buffer

PBP

parabrachial pigmented

PF

paraformaldehyde

PIF

paraintrafascicular

PN

paranigral

PV

parvalbumin

RLi

rostral linear nucleus of the raphe

TH

tyrosine hydroxylase

VGaT

vesicular GABA transporter

VGluT2

vesicular glutamate transporter type 2

VTA

ventral tegmental area

Footnotes

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

DATA ACCESSIBILITY

Supporting data are available.

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