Article Figures & Data
Figures
- Figure 1.
Localization of recorded GABA neurons in the tVTA. A, B, Light microscopy photographs showing staining for GAD (Ai, low-power view; Aii, high-power of area in red box in Ai) and μ receptors (Bi, low-power view; Bii, high-power of area in red box in Bi) in coronal slices through the tVTA. Staining for GAD or μ receptors highlights the tVTA (outlined with black dashed lines in A and B). C, Fluorescence microscopy photographs of staining for neurons (revealed by Fox3, green), GAD (red), and merged images in the VTA (top row) and tVTA (bottom row). Arrows indicate examples of GABA (GAD+) neurons. Note that the tVTA contains more GABA neurons than the VTA. D, Bar graph illustrating the proportion of GABA neurons throughout the rostrocaudal extent of the VTA–tVTA (plotted as proportion of Fox3+ neurons that are also GAD+; n = 3 rats, 6 hemispheres). The x-axis shows approximate anteroposterior distance from bregma (in millimeters). The red shaded area denotes tVTA as defined by Kaufling et al. (2009). The red dashed line defines the tVTA recording area used here, in which >75% of the neurons are GABAergic (GAD+). Ei, Typical example of average spike waveform of putative DA and GABA neurons recorded in the tVTA. Putative GABA-like spike waveforms are shorter than for putative DA neurons. Eii, Scatter plot illustrates the width of the first part of the action potential (AP) as a function of AP total duration for all tVTA recorded neurons. Two populations are clearly distinguishable. Classical AP waveforms for DA neurons are shown in blue (first part AP duration ≥1.1 ms and AP total duration ≥2.5 ms). Typical GABA-like waveforms are plotted in red and gray (corresponding to cells recorded in naive, PW, or 14DW rats). Note that chronic morphine treatment did not affect the AP duration of tVTA GABA neurons recorded (F(3,269) = 2.02, ns). F, High-power fluorescence photographs of immunohistochemistry for GAD (green) and μ receptors (red) in the tVTA. The merged image (GAD/μ) shows that GABA neurons express μ receptors (examples at arrows). Scale bars: Ai, Bi, 1 mm; Aii, Bii, 500 μm; C, 15 μm; F, 100 μm.
- Figure 2.
The firing rate of tVTA GABA neurons is lower in MD and PW animals than in naive rats. A, Schematics showing the localization of each tVTA GABA-like neuron recorded. Landmarks and position from bregma in millimeters (adapted from Paxinos and Watson, 2007). B, Photograph of a frontal section (neutral red stain) showing pontamine sky blue deposits made at the top and the bottom of a tVTA recording electrode track. Scale bar, 1 mm. C, Inset, Typical example of tVTA GABA-like neuron waveform. The width of the first waveform component was measured from the start of the spike to the negative trough, which was 0.65 ms in this example. Whisker plots show that the width of the first waveform component for putative GABAergic neurons were similar in all treatment groups.D, Bar graph showing the basal firing rates of tVTA GABA-like neurons in different treatment groups. Basal firing rates in MD and 14DW rats were lower than in naive/placebo rats. Intravenous NAL (0.1 mg/kg) in dependent rats (PW rats) normalized the basal firing rate. For all figures: naive/placebo rats, n = 60 cells, 20 rats; MD rats, n = 91 cells, 32 rats; PW rats, n = 53 cells, 23 rats; 14DW rats, n = 70 cells, 16 rats). **p < 0.01; ***p < 0.001. CLi, caudal linear nucleus of the raphe; cp, cerebral peduncle; IP, interpeduncular nucleus; ml, medial lemniscus; Pn, pontine nuclei; SNR, substantia nigra pars reticulata.
- Figure 3.
The firing of VTA DA neurons is the same in naive and PW rats. A, Photograph showing a pontamine sky blue deposit that marks a recording site in the VTA. Scale bar, 1 mm. B, Bar graph showing that the mean basal firing rate of VTA DA neurons in naive and 14DW rats is similar. C, Analyses of VTA DA neuron busting activity. Bars graphs show that bursting activity is similar between VTA DA neurons in naive and 14DW rats. For all figures: naive/placebo rats, n = 141 cells, 39 rats; 14DW rats, n = 114 cells, 25 rats. cp, cerebral peduncle; fr, fasciculus retroflexus; ml, medial lemniscus; mp, mammillary peduncle; %SIB, percentage of spikes in bursts; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata.
- Figure 4.
Intravenous morphine modulates tVTA GABA neurons in naive, MD, and acute withdrawal rats but has no effect on VTA DA neurons during withdrawal. A, Bar graph and schematics illustrating the effect of intravenous morphine (MOR; 1 mg/kg) on VTA DA and tVTA GABA neuronal activity during morphine treatment. Morphine activates DA VTA neurons in naive but not in 14DW rats (blue bars). Conversely, morphine inhibits tVTA GABA neurons in naive as well as in MD and 14DW rats, with no differences between these groups. VTA DA recording: naive rats, n = 7; 14DW rats, n = 6; tVTA GABA recording: naive rats, n = 6; MD rats, n = 6; 14DW rats, n = 6. B, Example traces (in blue) and firing rate histograms (in dark gray) from a naive VTA DA neuron (left) and a 14DW VTA DA neuron (right). The examples show the typical increased firing activity after morphine injection in naive but not in 14DW rat neurons. C, Example traces (in blue) and firing rate histogram (in dark gray) from tVTA GABA neurons recorded in a naive rat (left) or a 14DW tVTA rat (right) showing decreased firing activity after morphine injection in both cases. *p < 0.05.
- Figure 5.
Local μ receptor agonist inhibits tVTA GABA neurons in naive, MD, and PW rats. A, Bar graph reveals that local microinjections of DAMGO inhibit tVTA GABA neurons in naive, MD, and 14DW rats. Note that vehicle injections (ACSF) do not modify neuronal activity. ACSF injections: n = 9 cells, 4 rats (3 naive, 1 MD); DAMGO injection: naive rats, n = 10 cells, 7 rats; MD rats, n = 9 cells, 4 rats; 14DW rats, n = 11 cells, 5 rats. B, Examples of raw spike traces (in red) and firing rate histograms (in black) from two tVTA GABA neurons recorded in MD rats showing decreased firing activity after DAMGO injection (left) and the lack of effect after vehicle injection (right). *p < 0.05.
- Figure 6.
tVTA optogenetic activation inhibits VTA DA-like neurons in naive and PW rats. A, Schematics illustrate the virus injection protocol (left) and the photostimulation recording protocol (right). VTA DA neurons recorded during tVTA optogenetic stimulation. B, Fluorescence photographs of VTA sagittal sections showing immunofluorescent staining for eYFP (tag to mark ChR2 expression; i), TH (ii), and merge (TH/eYFP; iii), showing ChR2+ tVTA afferent fibers around VTA DA neurons after a tVTA virus injection. Scale bar, 1 mm. C, Fluorescence photographs of photomontaged sagittal sections showing the tVTA and VTA. Immunofluorescent staining for TH (i), eYFP (ii), and merge (iii). Note that the virus-infected area (green) is restricted to the tVTA region. There is no contamination of VTA DA neurons. Scale bar, 500 μm. D, Fluorescence photomicrographs of tVTA sagittal sections showing immunofluorescent staining for eYFP (i), GAD (ii), and merge (iii), showing tVTA GABA neurons that express ChR2 (white arrows). Scale bar, 1 mm. E, Pie charts represent the fraction of VTA DA recorded neurons responding to tVTA photostimulation in naive and 14DW rats that express ChR2 in tVTA. Note that the proportion of DA VTA responding neurons, ∼60%, is similar in the two groups. Naive rats, 23 of 41 inhibited cells; 14DW rats, 29 of 49 inhibited cells. F, Bar graphs illustrating latencies (i) and durations (ii) for VTA DA neurons in naive and 14DW rats inhibited at least 35% after photostimulation of ChR2-expressing tVTA neurons. There were no significant differences between groups. iii, PSTH illustrating a typical example from a naive rat of a VTA DA neuron inhibited by tVTA photostimulation (at time 0).
- Figure 7.
Expression of ChR2 in the tVTA for optogenetic experiments. A, Virus expression sites are plotted on four frontal sections from a rat brain atlas (Paxinos and Watson, 2007). Each viral site (n = 18) is presented at the level of its maximal extent. Approximate anteroposterior distance to bregma (in millimeters) is indicated on each drawing. B, Four examples of ChR2 expression in the tVTA. ChR2 expression was revealed using eYFP histochemistry. TH staining was added as a counterstain. CLi, caudal linear nucleus of the raphe; cp, cerebral peduncle; IP, interpeduncular nucleus; ml, medial lemniscus; SNR, substantia nigra pars reticulata.
- Figure 8.
ChR2 stimulation excites tVTA GABA neurons. A, Table showing the effect of tVTA optogenetic stimulation on tVTA GABA neurons. As expected, tVTA putative GABA neurons are stimulated by the tVTA stimulation. n = 6 cells, 2 naive rats. B, PSTH illustrating a typical example of tVTA short-latency responses to local tVTA photostimulation (at time 0).
- Figure 9.
Inactivation of tVTA neurons activates VTA DA-like neurons in naive but not in withdrawal rats. Ai, Schematic illustrates local MusB microinjection (0.8 mm, 500 nl) during VTA DA neural recording. Aii, Photograph of a frontal section through the tVTA showing an example MusB injection. Scale bar, 1 mm. B, Bar graph reveals the increase of activity in VTA DA-like neurons in naive but not in 14DW rats after tVTA MusB injection. C, Example trace (top) and firing rate histogram (bottom) from a naive VTA DA neuron showing the typical increased firing activity after tVTA MusB microinjection. D, Analysis of VTA DA neuron busting activity parameters. Bar graphs show that bursting in VTA DA-like neurons is increased after tVTA MusB injection in naive but not in 14DW rats. For all graphs: without MusB, n = 255 cells, 64 rats; after MusB: naive rats, n = 13 cells, 7 rats; 14DW rats, n = 19 cells/5 rats. *p < 0.05. cp, cerebral peduncle; IP, interpeduncular nucleus; ml, medial lemniscus; Pn, pontine nuclei; tth, trigeminothalamic tract; tVTA, tail of the ventral tegmental area; xscp, decussation of the superior cerebellar peduncle.
- Figure 10.
Local glutamate antagonist reveals a reduction of glutamatergic tone in the VTA of withdrawal rats. A, Bar graph illustrates that activity of VTA DA-like neurons is reduced by local CNQX/AP-5 microinjection in naive rats more than in 14DW rats. Note that vehicle (acsf) injections did not modify neuronal activity. ACSF, 19 cells, 4 naive rats; CNQX/AP-5 naive rats, n = 12 cells, 3 rats; 14DW rats, n = 21 cells, 3 rats. B, Examples of traces (top) and firing rate histograms (bottom) from two VTA DA-like neurons recorded in naive rats showing the typical decrease in activity after CNQX/AP-5 injection (left) and the lack of effect after vehicle injection (right). *p < 0.05.
