The lateral habenula (LHb) is involved in reward, aversion, addiction and depression through descending interactions with several brain structures, including the ventral tegmental area (VTA). The VTA provides reciprocal inputs to LHb, but their actions are unclear. Here we show that the majority of rat and mouse VTA neurons innervating LHb coexpress markers for both glutamate signaling (vesicular glutamate transporter 2; VGluT2) and GABA signaling (glutamic acid decarboxylase; GAD, and vesicular GABA transporter; VGaT). A single axon from these mesohabenular neurons coexpresses VGluT2 protein and VGaT protein and, surprisingly, establishes symmetric and asymmetric synapses on LHb neurons. In LHb slices, light activation of mesohabenular fibers expressing channelrhodopsin2 driven by VGluT2 (Slc17a6) or VGaT (Slc32a1) promoters elicits release of both glutamate and GABA onto single LHb neurons. In vivo light activation of mesohabenular terminals inhibits or excites LHb neurons. Our findings reveal an unanticipated type of VTA neuron that cotransmits glutamate and GABA and provides the majority of mesohabenular inputs.
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We thank R. Wise, J. Qi and J. Aceves for comments and J. Qi for assistance with viral injections used in ultrastructural experiments. Viruses were packaged by the NIDA IRP Optogenetics and Transgenic Technology Core. The Intramural Research Program of the National Institute on Drug Abuse, US National Institutes of Health (IRP/NIDA/NIH) supported this work.
The authors declare no competing financial interests.
Integrated supplementary information
(a) Representative FluoroGold (FG) injection site within the LHb. MHb - medial habenula. (b) Outlines of injection sites corresponding to 5 rats. Numbers refer to bregma level. Green = rat 1, purple = rat 2, blue = rat 3, orange = rat 4, red = rat 5. (c,d) VTA coronal section showing FG-labeled neurons (c, brown neurons under brightfield) and TH immunolabeled neurons (d, green fluorescence). (e-i) Higher magnification of boxed area in c. Mesohabenular neurons analyzed for VGluT2 mRNA and TH-immunofluorescent expression are indicated by different outlines: VGluT2+ TH– expression (solid green), VGluT2+ TH+ dual expression (dotted green), or lack of both VGluT2– and TH– expression (solid white). The majority of FG-labeled mesohabenular neurons (white in e, brown in g) lacks TH– (green in f), but expresses VGluT2+ mRNA (clusters of green grains in h, clusters of white grains in i). The second largest population of FG-labeled mesohabenular neurons co-expresses TH immunofluorescence and VGluT2 mRNA. (j) Frequency of mesohabenular neurons expressing VGluT2 mRNA or TH immunoreactivity (mean ± s.e.m). FG-VTA cell counting was made in 9 or 10 sections, each from rats #1, #2, and #3 (n = 3 rats), between bregma –4.9 mm and –6.1 mm.
(a,b) VTA coronal section showing FG-labeled neurons (a, brown neurons under brightfield) and GAD-expressing neurons (b, GAD mRNA expression; clusters of green grains). (c–e, f–h). Higher magnification of boxed areas in a. Mesohabenular neurons analyzed for GAD mRNA expression are indicated by different outlines: GAD expressing neurons [GAD+, solid red] and neurons lacking GAD expression [GAD–, solid white]. The majority of FG-labeled mesohabenular neurons (brown in c and f) express GAD mRNA (clusters of green grains in d and g, clusters of white grains in e and h). (i). Frequency of mesohabenular neurons expressing GAD mRNA (mean ± s.e.m). FG-VTA cell counting was made in 10 sections, each from rats #2, #3, and #4 (n = 3 rats), between bregma –4.9 mm and –6.1 mm.
(a) VTA coronal section from a rat injected with FG (see in Figure 1b,c). TH-immunofluorescence (green). Dotted yellow lines indicate VTA subregional boundaries. (b–g, h–m, and n–s). Higher power of boxed areas in a. Distinct phenotypes of FG-labeled mesohabenular neurons are indicated by different outlines: VGluT2+ GAD+ TH– (solid pink), VGluT2+ GAD– TH+ (dotted green), VGluT2– GAD+ TH– (solid red), VGluT2– GAD+ TH+ (dotted brown). In each panel, the most encountered mesohabenular neuronal phenotype, VGluT2+ GAD+ TH–, is displayed. (b–g) A FG-labeled VGluT2+ GAD– TH+ mesohabenular neuron (white in b, brown in e) expresses TH (green in c), lacks GAD mRNA (purple in d), but expresses VGluT2 mRNA (clusters of green grains in f, clusters of white grains in g). (h–m) A FG-labeled VGluT2– GAD+ TH– mesohabenular neuron (white in h, brown in k) lacks TH (green in i), expresses GAD mRNA (purple in j) and lacks VGluT2 mRNA (clusters of green grains in l, clusters of white grains in m). (n–s) A FG-labeled VGluT2– GAD+ TH+ mesohabenular neuron (white in n, brown in q) expresses TH (green in o), expresses GAD mRNA (purple in p) but lacks VGluT2 mRNA (clusters of green grains in r, clusters of white grains in s).
Circles represent one mesohabenular neuron from rats #2, #3, and #5 within sections between bregma –5.16 mm and –5.52 mm. Mesohabenular neurons were typically intermingled within a specific location of the anteromedial VTA (medial to the fasciculus retroflexus and around the borders of the rostral linear, interfascicular, medial paranigral, and medial parabrachial pigmented nuclei). (a–d) Left: Major phenotypes of mesohabenular neurons. The most frequently encountered mesohabenular neuronal phenotype dually co-expressed VGluT2 mRNA and GAD mRNA without TH immunoreactivity (pink). The second most frequently encountered mesohabenular neuronal phenotype triple co-expresses VGluT2 mRNA, GAD mRNA and TH immunoreactivity (purple). Right: Minor phenotypes of mesohabenular neurons. Small numbers of FG-labeled mesohabenular neurons express single VGluT2 mRNA (light green), dual VGluT2 and TH signals (dark green), single GAD mRNA (light red), dual GAD and TH signals (brown), single TH (blue), or were undetected with VGluT2, GAD, and TH (white). PIF = parainterfascicular nucleus; mp = mammillary peduncle; mtg = mammillotegmental tract.
Lateral habenula samples were processed for detection of VGluT2 by immunoperoxidase labeling (scattered dark material) and for detection of VGaT by immunogold (punctate dark circles, blue arrowhead in b). Experiments were repeated successfully three times. (a) At low magnification, three phenotypes of axon terminals (AT) are seen in the LHb. (b) A single AT co-expressing VGluT2 and VGaT forming both asymmetric (black arrow) and symmetric synapses (blue arrow) with dendrites. (c) AT expressing only VGluT2 without VGaT forming an asymmetric synapse (black arrow). (d) AT expressing only VGaT without VGluT2 forming two symmetric synapses (blue arrows). Axon terminals are outlined by pink, green, or blue dots. LHb dendrites are outlined by white dots. (e) Frequency of lateral habenula axon terminal phenotypes expressing VGluT2 alone, VGaT alone, or co-expressing VGluT2 and VGaT (mean ± s.e.m). Axon terminal counting was made from four Sprague-Dawley rats between bregma –3.0 mm and –4.2 mm.
Supplementary Figure 6 A single presynaptic mesohabenular terminal containing VGluT2 establishes multiple synapses.
(a–d) Four sequential serial sections of LHb from a VGluT2::Cre mouse expressing Cre-inducible mCherry following injection of an AAV-DIO-ChR2-mCherry vector in VTA. Detection of mCherry by immunoperoxidase labeling (scattered dark material) and detection of VGluT2 by immunogold (black dots indicated by black arrowhead in a). AT co-expressing mCherry and VGluT2 that establishes multiple asymmetric synapses (black arrows) with dendrite (De) or spine (sp). Mesohabenular axon terminals are outlined by green dots. LHb dendrites or spines are outlined by white dots. Experiments were repeated successfully three times.
Supplementary Figure 7 Single presynaptic mesohabenular terminals dually establish asymmetric and symmetric synapses.
(a–f) Six sequential serial sections in LHb from a VGluT2::Cre mouse delivered with a Cre-inducible AAV-DIO-ChR2-mCherry vector into the VTA. Tissue were processed for detection of mCherry by immunoperoxidase labeling (scattered dark material) and for detection of VGaT by immunogold (punctate dark circles, blue arrowhead in a). This axon terminal (AT) co-expresses mCherry and VGaT, establishes dual asymmetric (black arrows) and symmetric synapses (blue arrows) with dendrites (De), and puncta adhaerentia (white arrow). Mesohabenular axon terminals are outlined by red dots. LHb dendrites are outlined by white dots. Experiments were repeated successfully three times.
(a) Individual trials obtained from a VGluT2-ChR2 mouse. Upper traces show light-evoked inward currents obtained at –70 mV, lower traces show light-evoked outward currents obtained at –50 mV. Individual responses are shown in gray, averaged responses are shown in color. Dashed line indicates the onset of the laser pulse (5 ms). (b) Latencies and jitter (mean ± s.e.m) for all recorded neurons from rats and mice (n = 23 neurons). No significant differences in latency (paired t-test, t(22) = 1.733, p = 0.0971) or jitter were observed (paired t-test, t(22) = 0.3061, p = 0.7624). (c) Traces and time course from a single LHb neuron from a rat injected with a AAV-CAMKIIα-ChR2-eYFP vector in the VTA. Light-evoked inward and outward currents were observed at –70 mV and –50 mV, respectively. Application of TTX (1µM) eliminated both currents; subsequent application of 4-AP (200 µM) restored both inward and outward responses. (d) Summary of effects of TTX and TTX/4AP on percent of light-evoked responses. Left: CAMKIIα-ChR2 rats (F(1,6) = 46.2, p = 0.0005; *p = 0.0231, **p = 0.0013 versus TTX, two-way repeated measures ANOVA and Sidak’s multiple comparison test, n = 6 from 3 rats). Right: VGluT2-ChR2 mice (F(1,8) = 43.54, p = 0.0002; **p = 0.0014, ***p=0.00095 versus TTX, two-way repeated measures ANOVA and Sidak’s multiple comparison test, n = 8 from 3 mice). Data are mean ± s.e.m.
Supplementary Figure 9 Single 10-ms light stimulation of VTA VGluT2-ChR2 neurons evokes burst firing.
(a) Delivery of a Cre-inducible AAV-DIO-ChR2-eYFP vector into the VTA of VGluT2::Cre mice. Transfected VTA VGluT2 neurons and processes expressing ChR2-eYFP are indicated by dark blue signal. (b) VTA VGluT2-ChR2 neurons were recorded in vivo. A glass electrode was glued to an optical fiber and single 1 or 10 ms light pulses (473 nm) were delivered every 2 sec. c, Light-evoked action potentials were detected by the peak of their rising waveforms. (d–f) Light-evoked responses of VTA VGluT2-ChR2 neurons. Neurons responding to 1 ms light pulse with a firing fidelity over 90% were considered to be VGluT2-ChR2 neurons. (d) Top: Representative VTA VGluT2-ChR2 neuron exhibiting one or two spikes following each 1 ms light pulse. Bottom: The same neuron showed burst firing (3-6 spikes) in response to light pulses of 10 ms duration. (e) Single 1 or 10 ms light pulses evoked spiking with high fidelity in VTA VGluT2-ChR2 neurons. (f) Single 10 ms light stimulation reliably evoked burst firing in VTA VGluT2-ChR2 neurons (y-axis mean number of spikes).
(a) LHb neurons responding to light stimulation of mesohabenular ChR2-eYFP fibers from VGluT2-ChR2 mice or VGaT-ChR2 mice are represented as circles or triangles, respectively. Initially inhibited neurons are shown in red and initially excited neurons are shown in black. (b) LHb optical activation of mesohabenular fibers in CaMKIIα-ChR2 rats initially inhibited some LHb neurons (red squares), and initially excited others (black squares).
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Root, D., Mejias-Aponte, C., Zhang, S. et al. Single rodent mesohabenular axons release glutamate and GABA. Nat Neurosci 17, 1543–1551 (2014). https://doi.org/10.1038/nn.3823
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