Genetic targeting

All procedures followed United Kingdom Home Office regulations and were approved by the Animal Welfare and Ethical Review Panel of the Francis Crick Institute. Mice were kept on a standard 12-h/12-h light/dark cycle and on standard mouse chow and water ad libitum. Adult male and female mice (at least 8-week old) were used for in vitro experiments. Adult male mice were used for behavioral experiments, which were performed during the dark phase. The following previously characterized and validated transgenic mouse lines (or their crosses) were used, where indicated: MCH::Cre mice27, GAD65::Cre mice28, GAD65::GFP mice29, orexin::GFP mice30. The GAD65::Cre mice were bred in homozygous (hom)-WT pairs with C57BL/6 mice; all other transgenic mice were bred in het-WT pairs with C57BL/6 mice. For brain surgeries, mice were anesthetized with isoflurane and injected with meloxicam (2 mg/kg of body weight, s.c.) for analgesia. After placement into a stereotaxic frame (David Kopf Instruments), a craniotomy was performed and a borosilicate glass pipette was used to inject viral vectors bilaterally into the LH. Two injections (each 75 nL) were made into the LH in each hemisphere (bregma: −1.30 mm, midline: ±1 mm, from brain surface: 5.20 mm and 5.25 mm). Before any manipulations, mice were allowed to recover from surgery for at least 1 week while single-housed. To target expression of the activity indicator GCaMP6s to MCH LH neurons, we used an AAV vector carrying the 0.9 kb preproMCH gene promoter31, AAV9.pMCH.GCaMP6s.hGH (1.78 × 1014 gc/mL; Vigene Biosciences). The specificity of GCaMP6s expression was confirmed by staining with MCH antibody (Fig. 1b, 93% specificity was observed by analysis of MCH immunoreactivity colocalisation in 426 GCaMP6s neurons from three brains). For optogenetic silencing of MCH LH or GAD65 LH neurons, we injected Cre-dependent AAV8.Flex-ArchT-GFP (4.6 × 1012 gc/ml; UNC Vector Core) into LH of the MCH::Cre or GAD65::Cre mice, respectively. For ChR-assisted circuit mapping, “FLEX switch” ChR2 constructs were injected into LH of the MCH::Cre or GAD65::Cre mice, as indicated. These constructs were either AAV1.EF1.flox.hChR2(H134R)-mCherry.WPRE.hGH (8.78 × 1012 gc/mL; UPenn Vector Core) or AAV1.EF1.DIO.hChR2(H134R)-YFP.WPRE.hGH (6.2 × 1012 gc/mL; UPenn Vector Core). Cre-dependent “DREADD” chemogenetic actuator hM3Dq was targeted to GAD65 LH neurons in GAD65::Cre mice by injecting the vector AAV8.hSvn-DIO-hm3D(Gq)-mCherry (2.2 × 1012 genome copies (gc)/mL; UNC Vector Core) into LH of the GAD65::Cre mice16.

Fiber photometry

After LH injection of MCH-promoter-driven GCaMP6s, alone in C57/Bl6 mice or in combination with the Cre-dependent activatory DREADD (hM3Dq) in GAD65::Cre mice, fiberoptic implants were stereotaxically installed with the fiber tip above the LH (1.35 mm caudal from bregma, 1.0 mm lateral from midline, and 5 mm ventral from brain surface) and fixed to the skull as in our previous work32,13. This method is estimated to capture fluorescence signals from within ≈500 μm of the fiber tip32. Fiber tip locations were verified in each mouse by examining slices with a visible fiber tract. During fiber photometry experiments32, the excitation mode was set to provide interleaved 405 nm and 470 nm excitation light pulses via LEDs33. Fluorescence emission produced by 405 nm excitation is not sensitive to calcium and thus provides a real-time control for motion artefacts33. Fluorescence signals were normalized to produce the plotted % ΔF/F values as follows: ΔF/F = 100 * (Fr − F)/F, where Fr is the raw signal and F is the mean of the first 10 s of trial. Before photometry recordings, mice were habituated to the recording chamber, the plugging in procedure, and (where relevant) i.p. injections. On the day of fiber photometry recordings, mice were given 10 min to adjust to the chamber before an object was introduced. During the next 1 h mice had time to familiarize themselves with the object, after which the object was removed (i.p. injections of CNO or saline where performed where relevant at this point). Thirty minutes after this, the familiar object was reintroduced and mice had free access to explore it while their brain signals were recorded, and head location was video-tracked (Ethovision XP, 15 frames/s). The novel object trial followed, by exchanging the familiar object for a novel object and allowing mice to freely explore the novel object while their brain signals were recorded, and head location was video-tracked. Object exploration bouts were detected by nose video-tracking (Ethovision XP), and their onset defined as the first frame when the mouse nose entered the object area (defined as a 3 cm-wide perimeter around the object). Choice of objects for familiar and novel trials were based on a crossover design to avoid any confounding factors due to differences in objects. To compare MCH::GCaMP6s LH calcium signals between mice, we selected the first 10 exploration bouts of each mouse for the novel and familiar objects, and used these data to derive averaged signal per mouse. During the first 10 entries the object was investigated more frequently if it was novel (Supplementary Fig. 2H), and since our aim was to define neural correlates of behavioral responses to novel objects, we chose the first 10 entries for analysis of MCH photometry signals.

Closed-loop neural optosilencing

Mice were bilaterally LH-injected with Cre-dependent ArchT (or, in control experiments, Cre-dependent GCaMP6s), and bilaterally implanted with intra-LH optical fibers using the coordinates and procedures as described above for fiber photometry. Three weeks after surgery, mice were handled and habituated to the recording arena before any procedure started. For experiments, a green laser (532 nm, LaserGlow) was connected to the bilateral fibre implants to yield ≈20 mW light power output at the fiber tip. Since photometry recordings showed an onset of increased MCH neuron activity before mice entered the object area (Fig. 1d), we paired bilateral LH laser illumination with times when mouse nose was <2 cm away from object area (i.e., within 5 cm perimeter from object). For control experiments investigating the effect of silencing GAD65 LH or MCH LH neurons on object exploration, mice were freely behaving for 10 min in an open field arena with two identical objects, and the peri-object area of one object (defined as above) was paired with the bilateral LH laser illumination to test for the GAD65 LH or MCH LH neuron effects on object exploration (Supplementary Fig. 2A–D). The propensities for self-paced object investigation of GAD65::Cre and MCH::Cre mice were investigated in control experiments and found to be similar (Supplementary Fig. 2I).

Object recognition memory tests

For object recognition memory tests15 (Figs. 2c, d; 4c–e), during the laser ON familiarisation, the bilateral LH laser illumination was triggered whenever the mouse entered the peri-object area (as defined above) of either object. No laser was applied during recognition trials. Laser OFF familiarisation was performed in the same mice with the same temporal contingencies as laser ON familiarisation, but with a new set of objects. After 1 h of retention interval, during which mice were returned to their home cages and no experimental manipulations were performed, the recognition trial (= trial 2) consisted of 10 min during which mice freely explored one object from the previous familiarisation trial (familiar object) and one novel object. Sets of novel and familiar objects were alternated between mice in a crossover design. For novel object recognition tests where MCH receptors were blocked with SNAP 94847 (Fig. 4c–e), mice were i.p. injected with SNAP or vehicle solutions 45 min before trial 1. In these experiments, a longer interval between familiarisation/acquisition and recognition trials was used (20 h), to ensure that MCH receptors were only blocked during memory acquisition, and that mice were unimpaired by SNAP during recognition tests.

Our aim was to specifically examine the effects of LH optosilencing on memory formation, independently of factors such as the duration of sensory exposure to objects during familiarisation/memorisation. Therefore, in laser ON and laser OFF familiarization trials, a constant cumulative exposure of mice to objects was imposed, by real-time video tracking of the cumulative object encounter time (time when the mouse nose was in the object area), and terminating all trials when the same cumulative object encounter time (30 s) was reached. This ensured that differences in object memory acquisition were not due to variation in initial object exposure between different mice or trials, or different optosilencing conditions that may otherwise have influenced the total object investigation time as suggested by our control experiments (Supplementary Fig. 2A–D).

Y maze test of spatial memory

Continuous spontaneous alterations in a Y maze were measured with and without concurrent optogenetic silencing in the same mice (sequence of optogenetic silencing and laser off was alternated between mice) (Supplementary Fig. 4). Mice were connected to bilateral patch cords 10 min before start of the experiments and then transferred to the center of a standard Y maze (3 arms, 30 cm long, 120° apart). During the following 8 min, mice were free to explore the arms of the Y maze whilst video tracking with Noldus Ethovision scored the spontaneous alterations defined as consecutive entries into three different arms34,35.

Experimental sequences in behavioral experiments

Crossover-like experimental designs were used in all in vivo photometry and optogenetic experiments, to prevent artefacts and biases and isolate the effects of variables under investigation. Specifically, presentations of novel and familiar object were alternated within and between mice to avoid behavioral fatigue or order effects. Photometry experiments were designed to expose the same mouse to sequences of novel and familiar objects that avoided behavioral habituation or calcium indicator degradation as confounding factors (e.g., novel → familiar → novel, Fig. 1d, Supplementary Fig. 1B, C). Optogenetic experiments were based on a crossover-like design where manipulations involving drugs, laser light, or mouse genotype were arranged in a Latin square to avoid any confounding factors due to day to day differences or carry-over effects. To prevent potential arena side biases from influencing the results of experiments involving two objects positioned at different sides of arena, trials were repeated with laser OFF and ON sides reversed; the presented results are an average of both trials.

Channelrhodopsin-assisted circuit mapping in brain slices

For brain slice patch-clamp recordings combined with optogenetics16,36, LH slices were prepared at least 2 months after virus injection. Coronal brain slices containing the LH were cut at 250 μm thickness while immersed in ice-cold slicing solution. Slices were incubated for 1 h in artificial cerebrospinal fluid (ACSF) at 35 °C, and then transferred to a submerged-type recording chamber. Neurons containing fluorescent markers were visualized with an Olympus BX61WI microscope with an oblique condenser and fluorescence filters. Excitation light was delivered from a LAMBDA DG-5 beam switcher (Sutter) with a xenon lamp and ET470/40 (for ChR2) or ET500/20 (for ArchT) bandpass filters. A 40× 0.8NA objective was used to deliver pulses of excitation light (∼10 mW/mm2, 1 ms for ChR2 activation, or 1 s for ArchT activation) around the recorded cell, and postsynaptic responses were recorded in voltage-clamp (for circuit mapping) or current-clamp (for confirmation of ArchT-mediated photinhibition). Functional ChR2 expression was confirmed by recording light-activated action potentials in the target cells (n = 3 cells per group, not shown). For testing LH output connections of GAD65 LH cells, we chose LH neurons based on their genetic markers (MCH::GFP, orexin::GFP, GAD65::GFP) without noting GAD65 axon location. However, GAD65 axons were dense and abundant everywhere in the LH16.

Chemicals and solutions

For brain slice recordings, ACSF and ice-cold slicing solution were gassed with 95% O 2 and 5% CO 2 , and contained the following: 125 mM NaCl ACSF, 2.5 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 1.2 mM NaH 2 PO 4 , 21 mM NaHCO 3 , 2 mM D-(+)-glucose, 0.1 mM Na+-pyruvate, and 0.4 mM ascorbic acid. The slicing solution contained 2.5 mM KCl, 1.3 mM NaH 2 PO·H 2 O, 26.0 mM NaHCO 3 , 213.3 mM sucrose, 10.0 mM D-(+)-glucose, 2.0 mM MgCl 2 , and 2.0 mM CaCl 2 . For standard whole-cell recordings, pipettes were filled with intracellular solution containing the following: 120 mM K-gluconate, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA, 4 mM K2ATP, 2 mM Na2ATP, 0.3 mM Na2GTP, and 2 mM MgCl 2 (pH 7.3) with KOH. Gabazine (3 μm) was used where indicated. For in vivo chemogenetic manipulations, CNO was injected i.p. at 0.5 mg/kg body weight in experiments involving hM3Dq. The MCH receptor antagonist SNAP 94847 hydrochloride was injected i.p. at 20 mg/kg body weight (based on ref. 37) after being dissolved in distilled water with 10% DMSO and 30 mg/ml (2-Hydroxypropyl)-β-cyclodextrin. All chemicals were from Sigma or Tocris Bioscience.

Immunohistochemistry

For the immunolabeling of MCH neurons, 50-μm cryosections of pMCH-dependent GCaMP6s injected C57B/l6 mice were stained for MCH with a rabbit antibody to MCH (H-070-47,1:2000, Phoenix Pharmaceuticals) as a primary antibody, and Alexa 555–conjugated donkey antibody to rabbit IgG (A-21244, 1:500, Invitrogen) as a secondary antibody. Slices were then imaged with an Olympus VS120 slide scanner microscope and double labelling of GCaMP with Alexa 555 was quantified with ImageJ.

Statistical analyses

Statistical tests and descriptive statistics were performed as specified in the figure legends. All experimental animals were included in the analyses (no pre-selection or exclusion). In each experimental dataset at the cellular level, each n was a different cell (no repeated trials from the same cell were used as n values) and cells from at least three mice were analyzed. Before performing parametric tests, data were assessed for normality with a D’Agostino–Pearson omnibus test or Kolmogorov–Smirnov test for small sample sizes. To compare interactions within normally distributed data with repeated measurements, repeated measures ANOVA was used, with multiple comparison tests where appropriate. All statistical tests are two tailed unless otherwise stated. All error bars indicate the standard error of the mean. Analysis was performed with GraphPad Prism and MATLAB (The MathWorks, Inc.).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.