1 INTRODUCTION

The laterodorsal tegmentum (LDT) has been classically associated with attention and REM sleep (Datta & Siwek, 1997; Dort et al., 2015; Redila et al., 2015; Thakkar et al., 1996), but recent evidence showed that this nucleus also plays a role in locomotion and in reward-related behaviors (Dautan, Souza, et al., 2016; Gut & Mena-Segovia, 2019; Lammel et al., 2012; Steidl & Veverka, 2015; Xiao et al., 2016).

The LDT contains populations of acetylcholine, glutamate, and GABA neurons (Luquin et al., 2018; Wang & Morales, 2009) that project to diverse areas of the brain, including the thalamus, ventral tegmental area (VTA), nucleus accumbens (NAc), among others (Cornwall et al., 1990; Holmstrand & Sesack, 2011; Luquin et al., 2018; Wang & Morales, 2009). This places the LDT in a privileged anatomical position to modulate diverse circuits in the brain, including the reward circuit. For long it is known that the LDT provides a regulatory input to the VTA (Lammel et al., 2012; Oakman et al., 1995; Watabe-Uchida et al., 2012; Woolf & Butcher, 1986). Specifically, the LDT provides asymmetric (excitatory type) inputs to VTA dopaminergic neurons that preferentially innervate the NAc (Omelchenko & Sesack, 2005). Additionally, LDT–VTA cholinergic terminals were found to synapse on VTA dopamine neurons that innervate the NAc (Omelchenko & Sesack, 2006). It has been proposed a divergent LDT influence on mesoaccumbens neurons that appears to excite dopaminergic cells and inhibit GABA neurons of the VTA (Omelchenko & Sesack, 2005, 2006). Indeed, previous reports have shown that electrical stimulation of the LDT increases NAc dopamine levels by activating VTA dopaminergic cells through both glutamatergic and cholinergic receptors (Forster & Blaha, 2000; Forster et al., 2002). Additionally, LDT activity is essential for VTA dopaminergic burst firing (Lodge & Grace, 2006), which is considered to be the functionally relevant signal that encodes reward or indicates incentive salience/motivation (Berridge & Robinson, 1998; Cooper, 2002; Grace & Bunney, 1984; Schultz, 1998). More recently, it has been shown that the LDT also sends direct projections to the NAc (Coimbra et al., 2019; Dautan et al., 2014; Dautan, Hacioğlu Bay, et al., 2016), further supporting the importance of this brain region for the reward circuitry modulation.

Since LDT neurons are involved in the fine tuning of the VTA dopaminergic activity, it is becoming increasingly evident that this region plays an important role in reinforcement. Indeed, optogenetic excitation of LDT–VTA cells results in the acquisition of conditioned place preference (CPP) in rodents (Dautan, Souza, et al., 2016; Lammel et al., 2012; Xiao et al., 2016) and reinforces lever pressing in rats (Coimbra et al., 2017; Steidl, O'Sullivan, et al., 2017; Steidl & Veverka, 2015), suggesting that LDT–VTA inputs convey positive/rewarding signals. Less is known about the specific role of these projections in motivation and in reward value.

In this work we provide evidence about the role of LDT–VTA inputs in different dimensions of reinforcement, by optogenetically activating and inhibiting these inputs in a wide range of behavioral tests. Besides confirming previous studies of LDT–VTA involvement in inducing place preference and increasing operant behavior, we provide novel evidence about the role of LDT–VTA inputs in motivational drive and in value encoding.

2 MATERIAL AND METHODS 2.1 Animals and treatments

Male Wistar Han rats were individually housed under standard laboratory conditions (light/dark cycle of 12 hr; 22℃); food and water ad libitum, with enrichment materials in each cage (cardboard tubes, nesting materials). At the start of the experiments, 2–3-month-old males were used for electrophysiological and behavioral experiments. A limitation of this study is that we did not use females, however, it is important to refer that previous work from our group showed no significant differences in behavioral performance in the two choice task or progressive ratio in both sexes (data not shown).

Two different experiments were performed: in one group of animals we assessed electrophysiological evoked activity in the VTA by LDT terminal activation (nChR2 = 9; nNpHR = 5); and a different group of animals was used for behavioral experiments (at the start of experiment: nYFP = 10, nChR2 = 14; nYFP = 10, nNpHR = 14). All manipulations were conducted in accordance with European Regulations (European Union Directive 2010/63/EU). Animal facilities and the people directly involved in animal experiments were certified by the Portuguese regulatory entity—Direção Geral de Alimentação e Veterinária (DGAV). All experimental procedures are authorized by DGAV under project #23432 (2013) and #19074 (2016).

2.2 Constructs and virus preparation

AAV5–EF1a–WGA–Cre–mCherry, AAV5–EF1a–DIO–hChR2–YFP, AAV5–EF1a–DIO–NpHR3.0–YFP, and AAV5–EF1a–DIO–YFP were obtained directly from the Gene Therapy Center Vector Core (UNC) center. AAV5 vector titers were 2.1–6.6 × 1012 virus molecules/ml as determined by dot blot.

2.3 Surgery and cannula implantation

Rats designated for behavioral experiments were anesthetized with 75 mg/kg ketamine (Imalgene, Merial) plus 0.5 mg/kg medetomidine (Dorbene, Cymedica). 0.5 μl of AAV5–EF1a–WGA–Cre–mCherry and AAV5–EF1a–DIO–hChR2–YFP were unilaterally injected into the VTA (coordinates from bregma, according to Paxinos and Watson (2007): −5.4 mm anteroposterior, +0.6 mm mediolateral, and −7.8 mm dorsoventral) and LDT (coordinates from bregma: −8.5 mm anteroposterior, +0.9 mm mediolateral, and −6.5 mm dorsoventral), respectively (ChR2 group) or 0.5 μl of AAV5–EF1a–WGA–Cre–mCherry into the VTA and AAV5–EF1a–DIO–NpHR3.0–YFP in the LDT (NpHR group). Another group of animals was injected with 0.5 μl of AAV5–EF1a–WGA–Cre–mCherry in the VTA, and in the LDT, with 0.5 μl AAV5–EF1a–DIO–YFP (YFP groups). Rats were then implanted with an optic fiber (200 μm core fiber optic; Thorlabs) with 2.5 mm stainless steel ferrule (Thorlabs) using the injection coordinates for the VTA (with the exception of dorsoventral: −7.7 mm) that were secured to the skull using 2.4 mm screws (Bilaney, Germany) and dental cement (C&B kit, Sun Medical). Rats were removed from the stereotaxic frame and sutured. Anesthesia was reverted by administration of atipamezole (1 mg/kg). After surgery animals were given anti-inflammatory (Carprofeno, 5 mg/kg) for 1 day, analgesic (butorphanol, 5 mg/kg) for 3 days, and were let to fully recover for 30 days before initiation of behavior, to allow viral expression. Optic fiber placement was confirmed for all animals after behavioral experiments (Figure S1a, at the start of experiment: nYFP = 10, nChR2 = 14; nYFP = 10, nNpHR = 14). Animals that were assigned for electrophysiological experiments were not implanted with an optic fiber.

2.4 Behavior

Experimental design with groups and number of animals is depicted in Figure S2. Number of animals for behavioral experiments varied, considering that animals who lose fiber implants were removed from the experiment. Number of animals for behavioral experiments are as follows: real-time place preference (RTPP) and CPP—nYFP = 10; nChR2 = 14; nYFP = 10; nNpHR = 14; Operant behavior (Two-choice, PR, Extinction)—nYFP = 10; nChR2 = 11; nYFP = 10; nNpHR = 11. Experiments comprising YFP and ChR2 groups were replicated three times. Experiments comprising YFP and NpHR groups were performed once, considering the representative number of animals.

2.5 Subjects and apparatus

Rats were placed and maintained on food restriction (≈7 g/day of standard laboratory chow) to maintain 90% free-feeding weight. Behavioral sessions were performed in operant chambers (Med Associates) containing a central magazine that provided access to 45 mg standard food pellets (F0021, Dustless Precision Pellets, Bio-Serve), two retractable levers located on each side of the magazine with cue lights above them. A 2.8 W, 100 mA house light positioned at the top center of the wall opposite to the magazine provided illumination. A computer equipped with Med-PC software (Med Associates) controlled the equipment and recorded the data.

2.6 Two-choice schedule of reinforcement

During instrumental training, rats are presented two illuminated levers, one on either side of the magazine. Presses on one lever (Laser + pellet delivery (stim+ lever)) leads to instrumental delivery of a pellet plus 4 s blue (473 nm—80 10 ms pulses at 20 Hz) or yellow (589 nm—constant light) laser stimulation at 10 mW, paired with a 4 s auditory cue. In contrast, pressing the other lever (stim− lever) delivered a single pellet paired with another 4 s auditory cue, but with no laser stimulation. For both levers, presses during the 4 s after pellet delivery have no further consequence. After 2 days of habituation, each daily session begins with a single lever presented alone to allow opportunity to earn its associated reward (either stim+ or stim−), after which the lever is retracted. Then, the alternative lever is presented by itself to allow opportunity to earn the other reward, to ensure that the rat sampled both reward outcomes. Finally, both levers together are extended for the remainder of the session (30 min total), allowing the rat to freely choose between the two levers and to earn respective rewards in any ratio (FR1, FR4, RR4, and RR6).

2.7 Progressive ratio

The progressive ratio (PR) test was performed for either the stim+ or stim− lever in separate sessions, repeating the same conditions as described above: after lever press requirement achieved, for stim+ lever, pellet delivery was coupled to optical stimulation (4 s): blue laser, 473 nm—80 10 ms pulses at 20 Hz; or yellow laser, 589 nm—constant light at 10 mW. For stim− lever, reward consisted of a single pellet with no stimulation. The order of test conditions is counterbalanced across animals and repeated for each animal with the other lever. The number of presses required to produce the next reward delivery increases after each reward, according to an exponential progression (PR schedule: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219, 268,...) derived from the formula PR = [5e(reward number * 0.2)] − 5 and rounded to the nearest integer. To determine whether any preference in responding is the result of increased workload, animals are given a FR1 session after PR, identical to the initial day of training.

2.8 Extinction of food in the two-choice schedule of reinforcement task

To conversely assess whether laser stimulation alone can maintain responding when the reward is discontinued, rats are given the opportunity to press the same levers but without pellet (pellet extinction). Each completed trial (RR4) on the stim+ lever results in the delivery of laser stimulation and the previously paired auditory cue but no pellet delivery. Each completed trial on the other lever (stim−—previously pellet alone) resulted in the delivery of its auditory cue.

2.9 Conditioned place preference

The CPP apparatus consisted of two compartments with different patterns, separated by a neutral area (Med Associates): a left chamber measuring 27.5 cm × 21 cm with black walls and a grid metal floor; a center chamber measuring 15.5 cm × 21 cm with gray walls and gray plastic floor; and a right chamber measuring 27.5 cm × 21 cm with white walls and a mesh metal floor. Rat location within the apparatus during each preference test was monitored using a computerized photo-beam system (Med Associates). Briefly, on day 1, individual rats were placed in the center chamber and allowed to freely explore the entire apparatus for 15 min (pre-test). On day 2, rats were confined to one of the side chambers for 30 min and paired with optical stimulation—ON side; in the second session, rats were confined to the other side chamber for 30 min with no stimulation—OFF side. Optical stimulation consisted of 80 pulses of 10 ms at 20 Hz, every 15 s for blue light and 4 s of constant light at 10 mW, every 15 s for yellow light. Conditioning sessions were counterbalanced between animals. On day 3, rats were allowed to freely explore the entire apparatus for 15 min (test day). Results are expressed as the ratio of preference in the ON chamber and total time spent on each side of the apparatus.

2.10 Real-time place preference

The RTPP test was performed in a custom-made black plastic arena (60 × 60 × 40 cm) comprised by two indistinguishable chambers for 15 min. One chamber was paired with either blue light stimulation of 10 ms pulses at 20 Hz or constant yellow light stimulation, during the entire period that the animal stayed in the stimulus-paired side. The choice of paired chamber was counterbalanced across rats. Animals were placed in the no-stimulation chamber at the start of the session and light stimulation started at every entry into the paired chamber. Animal activity was recorded using a video camera and time spent in each chamber was manually assessed. Results are presented as percentage of time spent in each chamber.

2.11 In vivo single cell electrophysiological recordings

Experimental design with groups and number of animals is depicted in Figure S2a.

Four weeks after viral injection, animals were submitted to a stereotaxic surgery for the placement of the optic fiber coupled with tungsten recording electrode. Animals were anesthetized with urethane (1.44 g/kg, Sigma). The total dose was administered in three separate intra-peritoneal injections, 15 min apart. Body temperature was maintained at ~37℃ with a homeothermic heat pad system (DC temperature controller, FHC, ME, USA). Adequate anesthesia was confirmed by observation of general muscle tone, by assessing withdrawal responses to noxious pinching and by whisker movement.

Recording electrode coupled with a fiber optic patch cable (Thorlabs) was placed in the following coordinates: VTA: −5.4 from bregma, 0.6 lateral from midline, −7.5 to −8.2 ventral to brain surface. A reference electrode was fixed in the skull, in contact with the dura. We recorded nine animals for the ChR2 group and five for the NpHR group, averaging nine cells per animal.

Extracellular neural activity from the VTA was recorded using a tungsten recording electrode (3–7 MW at 1 kHz), that was lowered in increments of 100 μm, from −7.5 to −8.2. Recordings were amplified and filtered by the Neurolog amplifier (NL900D, Digitimer Ltd, UK) (low-pass filter at 500 Hz and high-pass filter at 5 kHz).

Spontaneous activity of single neurons was recorded to establish baseline firing rate for at least 60 s, as averaged number of spikes that occur in a 60 s period. The DPSS 473 nm and 589 nm laser system (CNI), controlled by a stimulator (Master-8, AMPI), was used for intracranial light delivery and fiber optic output was pre-calibrated to 10 mW. Optical stimulation was performed for each detected single neuron and consisted of: Optical activation: ChR2 group, 80 pulses of 10 ms at 20 Hz of blue laser; Optical inhibition: NpHR group, 4 s of constant yellow light;

Spikes of a single neuron were discriminated, and data sampling was performed using a CED micro 1401 interface and SPIKE 2 software (Cambridge Electronic Design, Cambridge, UK).

Firing rate was established for the baseline, stimulation period, and post-stimulation period (60 s after the end of stimulation).

Neurons showing a firing rate increase or decrease by more than 20% from the mean frequency of the baseline period were considered as responsive. A criterion of change of firing rate 20% above or below average activity of the baseline was used, as previously reported by others and us (Benazzouz et al., 2000; Coimbra et al., 2017, 2019; Soares-Cunha et al., 2016, 2020). A heatmap of neuronal response (in percentage) was generated (Figure 1j,m), comprising the following periods: 2 s pre-stimulus (baseline), stimulus and 2 s post-stimulus activity, using 50 ms time bins. We classified single units in the VTA into three separate groups of putative neurons: putative dopamine (DA), putative GABA, and “other” neurons. This classification was based on firing rate and waveform duration (calculated from average spike waveform) (Totah et al., 2013; Ungless & Grace, 2012; Ungless et al., 2004). Cells presenting a firing rate <10.0 Hz and a duration of >1.5 ms were considered putative DAergic (pDAergic) neurons. If the firing rate was >10.0 Hz and waveform duration <1.5 ms, cells were assigned to putative GABAergic (pGABAergic) neuron group. Other single units were excluded from analysis (n = 7 cells). This group likely contains units from both DA and GABA groups. At the end of each electrophysiological experiment, all brains were collected and processed to identify recording region.

Optogenetic modulation of LDT–VTA projections alters VTA neuronal activity. (a) Strategy used for LDT–VTA projection optogenetic stimulation and electrophysiological recordings in the VTA. (b) Representative immunofluorescence showing eYFP (green) expression in the LDT and mCherry (red) in the VTA; scale bar = 1 mm. (c) Representative immunofluorescence showing YFP staining in the LDT and in terminals in the VTA; scale bar = 100 μm. (d) Representative immunofluorescence showing YFP and NeuN staining in the LDT; scale bar = 100 μm. NeuN immunoreactivity identifies all cells in the LDT. (e) Summary graph showing the proportion of LDT cells (identified by NeuN-red) that express either opsin, ChR2 or NpHR (identified by GFP-green). ChR2 (n = 5) and NpHR (n = 5) animals. (f) Electrode placement for cell recording in the VTA for ChR2 (n = 9) and NpHR (n = 5) animals. (g) VTA neurons were separated into putative dopaminergic cells (pDAergic) and putative GABAergic cells (pGABAergic). (h) Majority of recorded cell in the ChR2 group are dopaminergic neurons. The majority of dopaminergic cells neurons significantly increase firing rate, whereas the opposite is observed in GABAergic cells, in response to optical stimulation (80 pulses of 10 ms at 20 Hz) of LDT terminals (n = 89 neurons/9 rats). (i) Temporal activity (0.5 s bins) of VTA pDAergic (upper panel) and pGABAergic (bottom panel) cells in response to LDT optical stimulation. Full line trace represents mean frequency of recorded cells and SEM as error is represented as shading. (j) Heatmap representation of percentages of pDAergic (upper panel) and pGABAergic (bottom panel) cell responses in the VTA upon activation of LDT terminals. (k) The majority of recorded cell in the NpHR group is dopaminergic neurons. Most of pDAergic and pGABAergic cells neurons significantly decrease firing rate, in response to optical inhibition (4 s of continuous yellow laser) of LDT terminals (n = 34 neurons/5 rats). (l) Temporal activity (0.5 s bins) of VTA pDAergic (upper panel) and pGABAergic (bottom panel) cells in response to LDT terminal inhibition. Full line trace represents mean of recorded cells and SEM as error is represented in the shading. (m) Heatmap representation of percentages of pDAergic (upper panel) and pGABAergic (bottom panel) cell responses in the VTA upon inhibition of LDT terminals. Bars represent mean and error bars denote SEM. *p < 0.05

2.12 Immunofluorescence

Animals were anesthetized with pentobarbital (Eutasil, Lisbon, Portugal) and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde. Brains were removed and sectioned sagittally at a thickness of 50 μm, on a vibrating microtome (VT1000S, Leica, Germany). Sections were incubated with the primary antibody chicken anti-mCherry (1:1,000, HBT008-100, HenBiotech) and rabbit anti-GFP (1:1,000, ab290, Abcam), followed by appropriate secondary fluorescent antibodies (1:1,000, anti-chicken Alexa Fluor 594, A-11042, anti-rabbit Alexa Fluor 488, A-21206, Invitrogen).

For c-fos experiments, animals were anesthetized with pentobarbital (Eutasil) 90 min after initiation of the PR test (vide experimental design in Figure S2a), and transcardially perfused as described above. Coronal sections were incubated with one of the following primary antibodies: mouse anti-TH (1:1,000, MAB318, Millipore); rabbit anti-c-fos (1:1,000, Ab-5, Millipore), goat anti-ChAT (1:750, AB144P, Millipore), sheep anti-ChAT (1:750, ab18736, Abcam), mouse anti-dopamine D1 receptor (1:300, sc-33660, Santa Cruz Biotechnology), mouse anti-dopamine D2 receptor (1:400, sc-5303, Santa Cruz Biotechnology), followed by appropriate secondary fluorescent antibodies (1:1,000) (anti-goat Alexa Fluor 594, A-11058, anti-mouse Alexa Fluor 594, A32744, anti-sheep Alexa Fluor 647, A-21448, anti-rabbit Alexa Fluor 488, A-21206, all from Invitrogen). Sections were stained with 4′,6-diamidino-2-phenylindole (DAPI; 1 mg/ml, D1306, Invitrogen) and mounted using mounting media (Permafluor, Invitrogen).

Slices for selected brain regions were the following (according to Paxinos coordinates (Paxinos & Watson, 2007)): LDT—from bregma −8.4 to −9.1; VTA—from bregma −5.2 to −6.3; and NAc—from bregma +2.8 to +0.8.

Image acquisition was performed by confocal microscopy (Olympus FV1000, Olympus) under 20× magnification. Confocal images were analyzed using ImageJ. Sections were labeled and areas delimitated relative to bregma using landmarks and neuro-anatomical nomenclature as described in Paxinos and Watson (2007). Positive cells within the brain regions of interest were manually analyzed in a blind manner, the same five sections per region per animal were considered (animals that performed task for stim+ lever: nYFP = 5; nChR2 = 5; nYFP = 5; nNpHR = 5; animals that performed task for stim− lever: nYFP = 5; nChR2 = 5; nYFP = 5; nNpHR = 5). Estimation of cell density of c-fos positive cells and double positive cells—cell number divided by the corresponding areas—was obtained for each region. Medial and lateral VTA subregions were selected according to the anatomical location of distinct dopaminergic sub-populations, as previously described (Beier et al., 2015; Lammel et al., 2008, 2011; Yang et al., 2018). The medial VTA was considered as the region comprising the paranigral nucleus and interfascicular nucleus and the lateral VTA was defined as the lateral parabrachial pigmented nucleus and the medial lemniscus adjacent to the substantia nigra. In addition, we divided NAc into subregions, core, and shell (Aragona et al., 2008; Bassareo et al., 2002; Dreyer et al., 2016).

To quantify the percentage of LDT neurons transfected with ChR2 or NpHR, we used slices containing the LDT of animals from electrophysiological experiments, from the coordinates mentioned above (nChR2 = 5 animals; nNpHR = 5 animals). Coronal sections were incubated overnight with the following primary antibodies: mouse anti-NeuN (1:750, MAB377, Millipore) and rabbit anti-GFP (1:1,000, ab290, Abcam), followed by appropriate secondary fluorescent antibodies (1:1,000, anti-mouse Alexa Fluor 594, A32744, anti-rabbit Alexa Fluor 488, A-21206, Invitrogen). Image acquisition was performed as mentioned, by confocal microscopy (Olympus FV3000, Olympus) under 20× magnification and analyzed using ImageJ. Sections were labeled and areas delimitated in the same manner as above. Positive cells within the LDT were manually analyzed in a blind manner and the same five sections per animal were considered. Quantification of proportion of LDT neurons (as assessed by NeuN-positive cells) that expressed ChR2 or NpHR (GFP-positive cells) was obtained by dividing the number of GFP-positive cells by the number of NeuN-positive cells in the LDT.

2.13 Statistical analysis

Statistical analysis was performed in GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS Statistics v24.0 (IBM corp, USA). Parametric tests were used whenever Shapiro–Wilk normality test SW >0.05.

Statistical analysis between two groups was made using Student's t test. Non-parametric analysis (Mann–Whitney test) was used when normality of data was not assumed. Repeated measures two-way analysis of variance (ANOVA) was used to compare groups versus sessions, groups versus positive cell density. Bonferroni's post hoc multiple comparison test was used for group difference determination. Report of statistical values is described along with results.

We compared neuronal firing rate distributions (baseline vs. stimulation) in the VTA (dopamine and GABA identified cells) using the two-sample Kolmogorov–Smirnov test (0.05 s bins spanning from 2 s before laser stimulation, during laser stimulation of 4 s, through 2 s after laser stimulation).

Pearson's correlation was used to examine the relationship between recruited c-fos+ cells and breakpoint levels reached in the progressive ratio task and between recruited c-fos+ cells in VTA and NAc subregions. Results are presented as scatterplots distribution and mean ± SEM. Statistical significance was accepted for p < 0.05.

3 RESULTS 3.1 Optogenetic modulation of LDT terminals changes VTA firing rate

We used a combined viral approach to specifically target LDT direct inputs to the VTA. We injected an adeno-associated virus (AAV5) containing a WGA–Cre fusion construct (AAV–EF1a–DIO–WGA–Cre–mCherry) in the VTA, and a vector encoding a cre-dependent ChR2 (optical excitation) or NpHR (optical inhibition) in the LDT (AAV–EF1a–DIO–hChR2–eYFP – ChR2 group; AAV–EF1a–DIO–NpHR–eYFP – NpHR group). Control animals were injected with AAV–EF1a–DIO–eYFP in the LDT (YFP groups). The WGA–Cre fusion protein is retrogradely transported (Gradinaru et al., 2010; Xu & Südhof, 2013), inducing the expression of cre-dependent ChR2- or NpHR–YFP only in LDT neurons that directly project to the VTA (Figure 1a,b). We were able to observe YFP staining throughout soma and dendrites of LDT neurons and in terminals located in the VTA, next to mCherry-positive cells (Figure 1c). In order to attest for the efficacy of the viral approach, we quantified the proportion of LDT neurons that expressed YFP (Figure 1d,e; percentage of cells expressing NeuN and YFP). 36.7% of NeuN-positive LDT neurons expressed ChR2–YFP and 36.3% expressed NpHR–YFP, after viral injections into the VTA and LDT.

To evaluate the functionality of the approach, we performed single cell electrophysiological recordings in anesthetized rats (Figure 1f–m), while optically stimulating LDT terminals in the VTA as previously described (Coimbra et al., 2017). Cells in the VTA were divided according to waveform duration and firing rate (Figure 1g) (Coimbra et al., 2017; Totah et al., 2013; Ungless & Grace, 2012; Ungless et al., 2004). Activation of LDT terminals (80 10 ms-light-pulse trains delivered at 20 Hz) evoked excitatory responses in 79% (55 of 70 cells) of VTA putative dopaminergic (pDAergic) neurons and inhibitory responses in 71% (10 of 14 cells) of VTA putative GABAergic (pGABAergic) recorded neurons (Figure 1h; n = 89 total recorded cells; n = 9 rats). Optical activation induced a significant increase in the firing rate of VTA pDAergic neurons in comparison to baseline (Figure 1i, Kolmogorov–Smirnov test, two-tailed, D = 1.00, p = 0.0028). Firing rate of pGABAergic neurons decreased with activation of LDT terminals in comparison to baseline (Figure 1i, Kolmogorov–Smirnov test, two-tailed, D = 1.00, p = 0.0014). A heatmap of firing rates of pDAergic cells showed that majority of cells increased firing rate during optical stimulation whereas pGABAergic displayed a decrease (Figure 1j). The percentage of VTA cells that responded to LDT manipulation was quite high as described in other studies (Lammel et al., 2011; Lodge & Grace, 2006), although others showed a reduced percentage of responsive neurons (Dautan, Souza, et al., 2016; Fernandez et al., 2018; Xiao et al., 2016).

Regarding optical inhibition experiments, we observed that 4 s stimulation of LDT terminals in the VTA reduced activity in 89.7% (26 of 29 cells) of VTA pDAergic neurons and in 60% (3 of 5 cells) of VTA pGABAergic recorded neurons (Figure 1k; n = 34 total recorded cells; n = 5 rats). Analysis of distribution showed that optical inhibition of LDT–VTA projections decreased VTA pDAergic and pGABAergic neurons firing rate in comparison to baseline (Figure 1l, VTA pDAergic: Kolmogorov–Smirnov test, two-tailed, D = 0.7778, p = 0.0364; VTA pGABAergic: Kolmogorov–Smirnov test, two-tailed, D = 0.8889, p = 0.0140). A heatmap of firing rates of pDAergic cells showed that majority of cells decreased firing rate during optical stimulation (both pDAergic and pGABAergic), but returned to baseline activity after (Figure 1m).

3.2 Optogenetic activation of LDT–VTA inputs enhances preference for a laser-paired reward

To test the impact of LDT–VTA manipulation on behavior, we used the previously described viral approach (Figure 2a) and unilaterally activated these inputs in freely moving rats during a two-choice instrumental task (Figure 2b). Animals were trained to press two levers to get food pellets; one of the levers was arbitrarily selected to deliver the pellet with simultaneous LDT–VTA optogenetic stimulation (stim+; blue laser: 80 10 ms pulses at 20 Hz), whereas pressing the other lever delivered only the pellet (stim−) (Figure 2b). The effort to get the pellet was increased until a random ratio of six lever presses per reward.

Optogenetic activation of LDT terminals in the VTA increases motivation. (a) Strategy used for LDT–VTA projection optogenetic stimulation during behavior. (b) Schematic representation of the two-choice task. Pressing stim− lever yields one food pellet and pressing stim+ lever delivers one pellet + optical stimulation of LDT–VTA inputs (80 10 ms pulses at 20 Hz). (c) Time-course representation of the responses in ChR2 (n = 11) and YFP (n = 10) rats. Optogenetic activation of LDT–VTA terminals focuses responses for the lever associated with the laser-paired reward (stim+) over an otherwise equivalent food reward (stim−) in ChR2 animals, but not in control YFP group. (d) Rats were subjected to two PR sessions, one for each lever: in one session, animals are tested for the stim+ lever, and in the other session animals are tested for the stim− lever. We observe an increase in the breakpoint for stim+ lever in ChR2 animals, indicative of enhanced motivation. (e) Total number of rewards earned during progressive ratio of ChR2 and YFP rats. ChR2 animals worked to earn more rewards for the stim+ than the stim− lever or YFP animals. (f) In pellet extinction conditions, both groups decrease responses for both levers. (g) CPP and (i) RTPP paradigms, in which one chamber is associated with laser stimulation (ON side). (h) Difference of total time spent in the OFF and ON sides in YFP (n = 10) and ChR2 (n = 14) groups. (j) Representative tracks for a ChR2 and a YFP animal during the RTPP. (k) Percentage of time spent on the ON and OFF sides, showing preference for the side associated with stimulation. Error bars denote SEM. *p < 0.05; **p < 0.01; ***p < 0.001 (in green significance for comparison within ChR2 group and in black for comparison between ChR2 and YFP)

ChR2 rats progressively discriminate and prefer the stim+ lever throughout the acquisition stage (Figure 2c; RM two-way ANOVA, session: F(3.524, 133.9) = 101.3, p < 0.0001; group: F(3, 38) = 39.73, p < 0.0001), without altering lever pressing performance, as the total number of lever presses was similar between ChR2 and YFP animals (Figure S3a). At the end of the acquisition stage, ChR2 animals exhibited a 4.6:1 ratio of preference for the stim+ lever in comparison to stim− lever (Bonferroni post hoc t test, t(12.45) = 10.98, p < 0.001). YFP animals had no preference for either lever.

To further evaluate motivation to work for the laser-associated reward, animals were subjected to the progressive ratio task, in which effort increases throughout the session. ChR2 animals presented increased cumulative presses in the stim+ lever versus stim− lever session (Figure S3c; RM two-way ANOVA, session: F(1.572, 59.73) = 210.1, p < 0.0001; group: F(3, 38) = 4.881, p = 0.0057; Bonferroni's post hoc t test, t(17.78) = 4.703, p = 0.0011). This resulted in a significant effect of interaction between groups and session in the breakpoint (Figure 2d; F(1, 19) = 48.63, p < 0.001). ChR2 animals presented a higher breakpoint in the stim+ lever than the stim− lever (Bonferroni's post hoc t test, t(19) = 9.045, p < 0.001), that is also significantly higher when compared to stim+ in YFP animals (Bonferroni's post hoc t test, t(38) = 6.620, p < 0.0001). ChR2 animals earn more rewards on the session for stim+ lever than the session for the stim− lever or YFP animals (Figure 2e; RM two-way ANOVA, F(1, 19) = 14.80, p = 0.0011; Bonferroni's post hoc t test, session: t(19) = 6.090, p < 0.0001; group: t(38) = 3.020, p = 0.0090).

Thus, activation of LDT–VTA projections is sufficient to increase preference for an otherwise equal reward and enhance motivation to work for that reward.

To further evaluate whether stimulation itself was an independent reinforcer, animals went through an extinction period, where pressing either lever did not yield any reward, but stim+ lever still originated laser stimulation. After a reminder session of FR1 schedule, both ChR2 and YFP animals decreased lever pressing in reward extinction conditions (Figure 2f; RM two-way ANOVA, session: F(1.119, 42.51) = 171.1, p < 0.0001). This suggests that if previously paired with a reward, laser excitation of LDT–VTA inputs alone is ineffective in inducing preference.

3.3 Optogenetic activation of LDT–VTA terminals is sufficient for place preference

To further understand the reinforcing properties of LDT–VTA terminal stimulation, we tested whether optogenetic modulation of LDT–VTA inputs induced place preference. We performed CPP (non-contingent) and RTPP (contingent) tests, pairing one chamber of each apparatus to laser stimulation (Figure 2g–k). Activating LDT–VTA terminals elicited place preference as shown by the increase in difference of time spent on the ON versus the OFF side of the CPP chamber of ChR2 animals (Figure 2h; t test, t(17) = 5.604, p < 0.0388).

In the RTPP, similarly, two-way ANOVA analysis showed a significant difference in the interaction between groups and chamber side (Figure 2k; two-way ANOVA, F(1, 22) = 30.09, p < 0.001).

ChR2 animals spent more time in the laser-paired chamber when compared to the OFF side (Bonferroni's post hoc t test, t(22) = 8.793, p < 0.001); and compared to YFP animals (t(44) = 5.486, p < 0.001). In both tests, YFP animals presented no preference for any chamber. Collectively, these results suggest that activation of LDT–VTA neurons triggers positive reinforcement.

3.4 Optogenetic inhibition of LDT–VTA inputs decreases the value of a reward and motivational drive

We next performed LDT–VTA inhibition experiments using a similar strategy as before. We injected a cre-dependent halorhodopsin in the LDT (AAV5-Ef1a-DIO-NpHR-eYFP-WPRE-pA) and WGA-cre vector in the VTA (NpHR group; Figure 3a). In the two-choice task (Figure 3b), optogenetic inhibition of LDT–VTA terminals decreased preference for the stim+ lever in NpHR animals in comparison to stim− (Figure 3c; RM two-way ANOVA, session: F(1.664, 63.24) = 87.29, p < 0.0001; group: F(3, 38) = 29.17, p < 0.0001; Bonferroni's post hoc t test, t(10.10) = 5.409, p = 0.0017). This increase in preference for the stim− was attributed to an increase in the motivation for that lever, considering that the total lever presses on this task was not different between groups (Figure S3b). Motivational drive for either lever was again tested with the progressive ratio test (Figure 3d,e). Optical inhibition of LDT–VTA projections decreased motivation, since NpHR animals showed less cumulative presses for the stim+ lever (Figure S3d; RM two-way ANOVA, session: F(1.330, 50.56) = 265.6, p < 0.0001; group: F(3, 38) = 6.409, p = 0.0013). NpHR animals present a robust decrease in the breakpoint (Figure 3d; RM two-way ANOVA; F(1, 19) = 26.94, p < 0.0001; Bonferroni's post hoc t test, t(19) = 6.856, p < 0.0001), including when compared to YFP animals (Bonferroni's post hoc t test, t(38) = 2.941, p = 0.0111). In agreement, the number of rewards earned during the progressive ratio task of NpHR animals is significantly less on the session for stim+ lever than the session for the stim− lever and between NpHR and YFP animals (Figure 3e; RM two-way ANOVA, F(1, 19) = 15.39, p = 0.0009; Bonferroni's post hoc t test, session: t(19) = 5.865, p < 0.0001; group: t(38) = 3.082, p = 0.0076).

Optogenetic inhibition of LDT terminals in the VTA decreases motivation. (a) Strategy used for LDT–VTA projection optogenetic inhibition during behavior. (b) Schematic representation of the two-choice task. Pressing stim− lever yields one food pellet and pressing stim+ lever delivers one pellet + optical inhibition of LDT–VTA inputs (4 s of constant yellow laser at 10 mW). (c) Time-course representation of the responses in NpHR (n = 11) and YFP (n = 10) rats. Optogenetic inhibition of LDT–VTA terminals shifts preference for the non-stimulated lever (stim−) in NpHR animals, but no preference is observed in YFP group. (d) Decrease in breakpoint for stim+ lever in NpHR animals. (e) Total number of rewards earned during progressive ratio of NpHR and YFP rats. NpHR worked less to earn rewards for the stim+ lever. (f) In pellet extinction conditions, both groups decrease responses for both levers. (g) CPP and (i) RTPP paradigms, in which one chamber is associated with laser stimulation (ON side). (h) Difference of total time spent in the OFF and ON sides in YFP (n = 10) and NpHR (n = 14) groups. (j) Representative tracks for a NpHR and a YFP animal during the RTPP. (k) Percentage of time spent on the ON and OFF sides, showing preference for the side associated with no stimulation. Error bars denote SEM. *p < 0.05; **p < 0.01; ***p < 0.001 (in orange significance for comparison within NpHR group and in black for comparison between NpHR and YFP)

In reward extinction conditions, both groups decreased instrumental responding for both levers since the first trial of extinction conditions (Figure 3f; RM two-way ANOVA, session: F(1.073, 40.76) = 112.4, p < 0.0001).

In the CPP test (Figure 3g), optical inhibition of LDT–VTA inputs did not induce statistically significant place preference/avoidance for any of the chambers (Figure 3h; t test, t(22) = 1.859, p = 0.076). LDT–VTA optical inhibition caused decreased preference for the ON chamber in the RTPP (Figure 3i–k; two-way ANOVA, interaction: F(1, 22) = 31.74, p < 0.0001). NpHR animals spent less time in the laser-paired side of the chamber than the OFF side (Bonferroni's post hoc t test, t(22) = 8.324, p < 0.0001); and also in comparison to YFP animals (Bonferroni's post hoc t test, t(44) = 5.634, p < 0.0001).

3.5 Differential recruitment of VTA and NAc neuronal populations

We determined the activation pattern of different neuronal populations after the PR task in YFPs, ChR2, and NpHR groups, while optically manipulating LDT terminals in the VTA during the reward period of the PR test, in animals working for either stim− lever or stim+ lever (Figure S4). We next evaluated the density of positive c-fos+ cells that were either ChAT+ (cholinergic neurons) or ChAT− in the LDT (Figure 4a,b), TH+ or TH− in the VTA (Figure 4e,f), and D1R+, D2R+ or ChAT+ in the NAc (Figure 4j,k).

Recruitment of neurons in the LDT, VTA, and NAc after optical modulation of LDT–VTA terminals. (a) Representation of LDT visualized at bregma = −8.5 AP, scale bar = 250 μm. (b) Immunofluorescence of the LDT with staining for cell nuclei (DAPI, blue), ChAT (red), and c-fos (green), scale bar = 100 μm, inset of positive cell. (c) Density of c-fos and ChAT double positive cells in ChR2 and YFP rats or (d) NpHR and YFP after PR performance on the stim+ or stim− lever. (e) Representation of medial and lateral VTA subregions visualized at bregma = −5.2 AP, scale bar = 100 μm. (f) Immunofluorescence of the VTA, staining for cell nuclei (DAPI, blue), tyrosine hydroxylase (TH, red), and c-fos (green), scale bar = 100 μm, inset of positive cell. (g) Density of c-fos and TH double positive cells in ChR2 and YFP rats or (h) NpHR and YFP after PR performance on the stim+ or stim− lever. There is an increase in the number of c-fos+/TH+ cells in the lateral VTA after LDT–VTA optical activation, whereas optical inhibition shows no significant differences in the number of recruited cells when compared to YFP animals. (i) Representation of NAc core and shell subregions. (j) Immunofluorescence of the NAc, staining for cell nuclei (DAPI, blue), c-fos (green) and D1R (red), (k) DR2 (red) or (l) ChAT (red), scale bar = 100 μm. Inset of positive cells in each staining. (m) Density of c-fos and D1R, DR2 or ChAT double positive cells in ChR2 and YFP rats or (n) NpHR and YFP rats after PR performance on the stim+ or stim− lever. There is an increase in the number of c-fos+/D1R+ cells in both NAc core and shell subregions after LDT–VTA optical activation, whereas optical inhibition appears to recruit mostly D2R cells. No significant differences were found in the number of c-fos+/ChAT+ cells. For all cell countings: nstim+ = 5; nstim− = 5 in each group. Error bars denote SEM. **p < 0.01; ***p < 0.001

In the LDT, ChR2 stim+ group displayed an increase in c-fos+ cells in comparison to ChR2 stim− or YFP groups (Figure S4a; one-way ANOVA, F(3, 16) = 10.98, p = 0.0004; ChR2 stim+ vs. stim−: post hoc t test, t(16) = 5.506, p = 0.0003; ChR2 stim+ vs. YFP stim+: post hoc t test, t(16) = 4.002, p = 0.0062). In agreement, ChR2 stim+ group presented an increase in c-fos+/ChAT+ (Figure 4c; two-way ANOVA, F(3, 16) = 6.697, p = 0.0039) in comparison to ChR2 stim− (ChAT: post hoc t test, t(32) = 6.563, p < 0.0001), or in comparison to YFP animals stimulated animals (ChAT: post hoc t test, t(32) = 6.535, p < 0.0001). No effect was observed in c-fos+/ChAT− cells.

No significant differences were observed in c-fos+ cell density of NpHR or YFP-inhibition groups, independently of the lever (Figure S4b). Nevertheless, NpHR stim+ group showed a specific decrease in cell density of c-fos+ChAT+ in LDT, when compared to the NpHR stim− group (Figure 4d; two-way ANOVA, F(3, 16) = 14.28, p < 0.0001; post hoc t test, t(32) = 4.305, p = 0.0009). Recruitment of ChAT− cells was increased in NpHR stim+ animals when compared to NpHR stim− (Bonferroni's post hoc t test, t(32) = 5