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GABA/glutamate co-release controls habenula output and is modified by antidepressant treatment

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Science  19 Sep 2014:
Vol. 345, Issue 6203, pp. 1494-1498
DOI: 10.1126/science.1250469

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  1. Fig. 1 Mixed excitatory and inhibitory transmission from EP input controls persistent LHb activity.

    (A) Diagram of experimental protocol. Microscopic images depict example injection of AAV-ChR2-YFP into WT rat; left, yellow fluorescent protein (YFP) fluorescence; right, bright-field illumination. (B) Trains of synaptic responses evoked by light pulses (50 Hz, blue dots) before (top) and after (bottom) bath application of picrotoxin recorded in whole-cell current clamp mode. Scale bars, 25 mV, 50 ms. (C) Summary of average responses for recording in (B). Similar results were obtained in three cells. (D) Whole-cell recordings in voltage-clamp mode of light-evoked EP-LHb transmission at different holding potentials (as indicated) and after application of drugs (as indicated). Scale bars, 100 pA, 10 ms. (E) Current-voltage plots for different EP-LHb synaptic conductances normalized to AMPA-R–mediated response at –60 mV (N = 9 cells). (F) Cell-attached (top) and subsequent whole-cell (middle) recordings at +20 mV (GABA, gray) and –50 mV (AMPA, black) holding potentials from two cells (i and ii) during synaptic responses evoked by pairs of light pulses (as indicated). Below, plot of change in firing rate (measured in cell-attached mode) versus GABA-A-R–mediated/AMPA-R–mediated synaptic response (measured in voltage-clamp mode; N = 24 cells); values for cells i and ii indicated by gray symbols. Scale bars, 400 pA, 50 ms. Error bars throughout indicate mean ± SEM.

  2. Fig. 2 Co-release of GABA and glutamate from EP inputs to LHb: Electrophysiological, optogenetic, and mouse genetic evidence.

    (A) Whole-cell recording at –15 mV holding potential of light-evoked EP-LHb transmission after bath application of indicated drugs. Scale bars, 60 pA, 10 ms. (B) Spontaneous responses from whole-cell recordings at –15 mV holding potential from three different LHb neurons (i, ii, and iii) in adult WT mice. Lower trace (iii′) is the same as above (iii) at higher temporal resolution. Scale bars, 50 pA, 200 ms (upper), 10 ms (lower). Biphasic events accounted for 2/3 of all large (>15 pA) outward events in these cells (79, 56, and 67%; large, biphasic events were found in 3 of 12 recordings). (C) Spontaneous miniature biphasic events (*) from whole-cell recordings at –40 mV with 0.5 mM chloride internal solution. Scale bars, 10 pA, 20 ms. (D) Whole-cell recording from Vglut2-cre mouse injected with AAV-DIO-ChR2-YFP at –15 mV holding potential of light-evoked EP-LHb transmission with indicated drugs in bath. Both inward and outward currents found in 17 of 17 cells. Scale bars, 100 pA, 10 ms. (E) Plot of effect of light-evoked EP-LHb transmission from Vglut2+ axons of adult mice delivered at 50 Hz (left) or 2 Hz (right) on normalized LHb neuronal firing rate before and after bath application of picrotoxin (*P < 0.05). (F) Same as (D) for GAD67-cre mice. Total of 11 of 13 cells, inward/outward; 2 of 13 cells, outward only. Scale bars, 40 pA, 10 ms. (G) Cell-attached recording (average of 10 trials) from adult GAD67-cre mouse LHb neuron; light delivery indicated. Scale bars, 5 mV, 20 ms.

  3. Fig. 3 Co-localization of GABA-producing enzyme (GAD) and vesicular glutamate transporter (Vglut2) at EP neuron terminals in LHb.

    (A) Confocal fluorescent images of LHb sections from mouse injected with AAV-GFP into EP labeled for indicated proteins. Thick arrows indicate some of the sites of Vglut2, GAD, and GFP colocalization. Thin arrow indicates Vglut2 and GAD colocalization (with no GFP). Asterisk indicates GFP-expressing structure showing only Vglut2 expression. Scale bar, 3 μm. (B) Confocal fluorescent images of sections from LHb (top) and outside LHb (bottom) labeled for indicated proteins. Arrows indicate structures displaying co-labeling for GAD and Vglut2. Scale bar, 3 μm. (C) Distribution of GAD fluorescence in pixels with Vglut2 intensity above Vglut2 threshold [dashed line in (D); (26)], for images in LHb (for pixels displaying GFP, black line) and outside LHb (gray line). (D) Distribution of Vglut2 fluorescence in pixels with GAD intensity above GAD threshold (dashed line in C), as indicated. [(C) (D)] EP-LHb, N = 21 images, 3 mice; outside LHb, N = 5 images, 3 mice. (E) Electron micrograph showing a GFP-containing region in the LHb. Gold particles in the mitochondria were not analyzed. Scale bars, 200 nm. See figs. S17 and S18 for larger images of same region. (F) (Left) 18-nm gold labels Vglut2 (1); 12-nm gold labels GABA (2). (Middle) 18-nm gold labels Vglut2 (1); 6-nm gold labels GFP (3). (Right) 12-nm gold labels Vglut2 (1); 6-nm gold labels GABA (2). Scale bars, 30 nm. (G) Density of gold label pairs for distance intervals on x axis for a single experiment. Black line, Vglut2-GABA distance; gray line, GABA-GFP; dotted gray line, Vglut2-GFP. N = 72 Vglut2 (18 nm) labels, 486 GABA (12 nm) labels, 416 GFP (6 nm) labels. Arrow indicates peak for Vglut2-GABA counts at 20 to 30 nm. (H) Increased enrichment (26) of Vglut2-GABA (black lines; three experiments) but not GABA-GFP (gray lines; two experiments) or Vglut2-GFP (dotted gray; two experiments) labels at 15- to 30-nm separation. N = 1624, 2556, and 1558 total Vglut2, GABA, and GFP labels, respectively. *Values > 4 SD from the mean.

  4. Fig. 4 Altered co-release of GABA in conditions related to depression.

    (A) Sample whole-cell recordings of GABA-A-R- (black) and AMPA-R–mediated (gray) light-evoked EP-LHb responses from animals treated with saline or citalopram (10 mg per kg of weight, intraperitoneally). Scale bars, 200 pA, 20 ms. (B) Sample recordings, as in (A), in WT rats and cLH rats. Scale bars, 200 pA, 20 ms. (C) Bar graph of GABA-A-R–mediated/AMPA-R–mediated response at EP-LHb synapses for indicated groups normalized to average of saline ratios (N = 19 cells from four saline-treated mice and 24 cells from four citalopram-treated mice). (D) Bar graph of responses, as in (C), for WT and cLH rats normalized to average of WT ratios (N = 41 cells from nine WT rats and 23 cells from nine cLH rats). (E) Ratio of average background subtracted pixel intensities for GAD and Vglut2 in terminals originating from EP (26) for indicated groups (N = 24 images from three saline-treated mice, 21 images from three citalopram-treated mice). (F) Ratios, as in (E), for WT and cLH rats (N = 32 images from seven WT rats, 21 images from three cLH rats). (G) Example confocal images from a saline and a citalopram-treated mouse. Scale bar, 3 μm. (H) Example confocal images from a WT rat and a cLH rat. Scale bar, 3 μm. (I) GAD fluorescence in pixels displaying Vglut2 and GFP labels in tissue from animals treated with citalopram or saline (percentage of Vglut2+, GFP+ pixels showing GAD fluorescence: saline, 54 ± 5%, 21 images; citalopram, 79 ± 2%, 24 images; P < 10−4). (J) Vglut2 fluorescence in pixels displaying GAD and GFP labels in tissue from animals treated with citalopram or saline (percentage of GAD+, GFP+ pixels showing Vglut2 fluorescence: saline, 80 ± 3%, 21 images; citalopram, 79 ± 3%, 24 images; P = 0.8). (K) GAD fluorescence, as in (I), for images from WT and cLH rats (WT, 50 ± 3%, 32 images; cLH, 19 ± 3%, 21 images; P < 10−7). (L) Vglut2 fluorescence, as in (J), for images from WT and cLH rats (WT, 61 ± 3%, 32 images; cLH, 61 ± 3%, 21 images; P = 0.9). *P < 0.002.

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