Food cue regulation of AGRP hunger neurons guides learning – Nature

No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Experimental subjects

Vglut2-IRES-Cre (JAX 016963)³³, Vglut2-IRES2-FlpO (JAX 030212) (unpublished, donating investigator H. Zeng (Allen Institute for Brain Science)), AGRP-IRES-Cre (JAX 012899)³⁴, Pdyn-GFP³⁵ and wild-type mice (JAX101045) were obtained through Jackson Laboratories or in house. Lepr-IRES-Cre³⁶ mice were maintained as previously described¹². All mice were maintained on a mixed genetic background unless otherwise noted. Pdyn-IRES-Cre²⁷ mice were maintained as previously described and kept on a congenic C57 BL/6J background. The National Institute of Health and Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee approved all animal care and experimental procedures. Mice were housed at 22–24 °C, 20–30% humidity with a 12:12 light:dark cycle with standard mouse chow (Teklad F6 Rodent Diet 8664) and water was provided ad libitum, unless otherwise stated. All diets were provided as pellets. For all behavioural studies, we used male mice between 8 and 20 weeks of age. For electrophysiogical recordings, we used male mice between 8 and 12 weeks of age.

Brain tissue preparation

Mice were terminally anaesthetized with chloral hydrate (Sigma-Aldrich C8383) and transcardially perfused with phosphate-buffered saline (PBS) followed by 10% neutral buffered formalin (Fisher Scientific SF100). Brains were extracted then cryoprotected in 20% sucrose. All brains were sectioned coronally on a freezing sliding microtome (Leica Biosystems) at 40 μm and collected in four equal series.

Immunohistochemistry

Tissue sections were washed with 0.1 M phosphate-buffered saline (pH 7.4) then blocked in 5% normal donkey serum/0.2% Triton X-100 in PBS for 1 h at room temperature. Sections were then incubated overnight at room temperature in blocking solution containing: rat anti-mCherry (1:3,000, Invitrogen M11217) and chicken anti-GFP (1:1,000, Invitrogen A10262). Secondary detection was performed with Alexa Fluor 488 or 594 conjugated donkey anti-chicken or donkey anti-rat (1:1,000, Invitrogen) for 1 h at room temperature. After secondary incubation, sections were washed and mounted onto gelatin-coated slides and fluorescent images were obtained with an Olympus VS120 slide scanner microscope. Our inferred Bregma coordinates on all histological images were adopted from a stereotaxic atlas³⁷.

Stereotaxic surgeries and viral injections

For viral injections, six-to-eight week old male mice were anaesthetized with a ketamine (100 mg kg⁻¹) and xylazine (10 mg kg⁻¹) cocktail diluted in 0.9% saline and placed into a stereotaxic apparatus (Kopf model 940). Subcutaneous injection of sustained-release meloxicam (4 mg kg⁻¹) was provided as postoperative care. A pulled glass micropipette (20–40 μm diameter tip) was used for stereotaxic injections of AAV. For electrophysiological experiments, bilateral injections (25 nl) of purified AAV (6.24 × 10¹² viral genomes ml⁻¹) were injected into the NAc (from bregma: +1.3 AP, ± 0.5 ML, −4.25 DV), BNST (from bregma: +0.14 AP, ± 0.75ML, −4.9 DV), MPO (from bregma: +0.4 AP, ± 0.25 ML, −4.75 DV), LH (from bregma: −1.3 AP, ± 0.9 ML, −5.3 DV), VTA (from bregma: −3.15 AP, ± 0.6 ML, −4.74 DV), IPN (from bregma: −3.4 AP, ± 0.0 ML, −4.8 DV), and PAG (from bregma: −4.16 AP, ± 0.3 ML, −2.5 DV). For optogenetic experiments, bilateral injections (15 nl) of AAV9-EF1α-DIO-ChR2(H134R)-eYFP purchased from the University of Pennsylvania School of Medicine Vector Core (donating investigator, K. Deisseroth; AV-9-20298P; Addgene: 20298; 2.7 × 10¹³ viral genomes ml⁻¹) were injected into the LH (coordinates as above). For in vivo fibre photometry experiments, AAV1-hSyn-DIO-GCaMP6s (University of Pennsylvania Vector core; Addgene 100845-AAV1; 1.6 × 10¹³ viral genomes ml⁻¹) was injected unilaterally into either the LH (25 nl, coordinates as above), DMH (25 nl, from bregma: −1.80 AP, ± 0.3 ML, −5.2 DV), or ARC (150 nl, from bregma: −1.45 AP, ± 0.25 ML, −5.85 DV). For retrograde chemogenetic silencing studies, AAV6-CAG-FlpO (Boston Children’s Hospital Viral Vector Core, modified from Addgene 67829; 2.88 × 10¹⁴ viral genomes ml⁻¹) was bilaterally injected into the DMH (15 nl, coordinates as above) and AAV8-nEF-fDIO-hM4Di-mCherry (Boston Children’s Hospital Viral Vector Core, modified from Addgene 44362 and 55644; 9.98 × 10¹³ viral genomes ml⁻¹) was bilaterally injected into the LH (25 nl, coordinates as above). For Flp-dependent chemogenetic studies in combination with fibre photometry, AAV8-nEF-fDIO-hM4Di-mCherry was bilaterally injected into the LH (25 nl, coordinates as above) or, the DMH (25 nl, coordinates as above). For tetanus toxin mediated silencing studies, AAVDJ-CMV-DIO-eGFP-2A-TeNT (Stanford; GVVC-AAV-71; 3.6 × 10¹² viral genomes ml⁻¹) was injected bilaterally into the DMH (15 nl, coordinates as above). Finally, for projection specific tetanus toxin mediated silencing studies, AAVDJ-CMV-DIO-eGFP-2A-TeNT was injected bilaterally into the LH (20 nl, coordinates as above) and rAAV2-hSyn-Cre (Addgene 105553; 1.2 × 10¹³ viral genomes ml⁻¹) was injected bilaterally into the DMH (15 nl, coordinates as above). Mice were allowed to recover for a minimum of three weeks before the initiation of any experiments. Following each experimental procedure, accuracy of AAV injections were confirmed via post hoc histological analysis of mCherry, YFP, or GFP fluorescent protein reporters, viral expression of each individual surgery is catalogued in detail within Extended Data Figs. 7–11. All subjects determined to be surgical ‘misses’ were those with absent or low reporter expression and were removed from the experimental dataset. In addition, mice were excluded from the data set if reporter expression was primarily outside of the area of interest. Anatomical boundaries were drawn by using the DAPI signal so that we could clearly discern landmarks within a histological section. On the basis of the landmarks present, we then inferred the A/P coordinate in the atlas³⁷ and traced the outline of different nuclei and superimposed the outline on our histological images Extended Data Fig. 12.

Optic fibre implantation

Optic fibre implantations were performed during the same surgery as viral injections (above). For optogenetic photostimulation of LH → DMH terminals, ceramic ferrule (Precision Fibre Products) optical fibres (200 μm diameter core, 0.39 NA, multimode; Thorlabs) were implanted within the midline over the DMH (from bregma: −1.8 AP, ± 0 ML, −4.7 DV). For LH^VGLUT2 → DMH axon fibre photometry, a metal ferrule (Precision Fibre Products) optic fibre (400 μm diameter core, 0.5 NA, multimode; Thorlabs) was implanted unilaterally over the DMH (from bregma: −1.8 AP, ± 0.3 ML, −5.1 DV). For DMH^LEPR and ARC^AGRP cell body fibre photometry, a stainless steel ferrule optic fibre (same as above) was implanted unilaterally over the DMH (coordinates as above) or the ARC (from bregma: −1.45 AP, ± 0.25 ML, −5.8 DV). Fibres were fixed to the skull using dental acrylic and mice were allowed to recover for three weeks before the start of acclimation to behavioural testing.

Monosynaptic rabies mapping

Lepr-IRES-Cre mice were unilaterally injected with a 1:1 mixture of AAV8-EF1α-DIO-TVA-mCherry (University of North Carolina Vector Core, donating investigator N. Uchida) and AAV8-CAG-FLEX-Rabies G (Stanford; GVVC-AAV-59) into the DMH (15 nl, coordinates as above) (Extended Data Fig. 2a). Mice recovered for three weeks after TVA and RG transduction to ensure adequate levels of TVA and RG viral expression. Following recovery, mice underwent a second surgery in which mice were injected with SADΔG-EGFP (EnvA) rabies (Salk Gene Transfer Targeting and Therapeutics Core) into the DMH (15 nl, coordinates as above). Mice recovered for 7 days to allow for the retrograde transport of rabies virus and EGFP expression before perfusion and histological processing. Sites of afferent input to DMH^LEPR neurons were assessed by the presence of EnvA-eGFP positive neurons.

Rabies collateral mapping

Vglut2-IRES-Cre mice were unilaterally injected with AAV8-EF1α-DIO-TVA-mCherry into the LH (15 nl) and allowed to recover for three weeks (Extended Data Fig. 3e–g). Then, SADΔG-EGFP (EnvA) rabies was unilaterally injected into the DMH (15 nl). Mice were allowed to recover for seven days to allow for the retrograde transport of rabies virus and EGFP transgene expression before perfusion and histological processing. Comprehensive examination of SADΔG-EGFP (EnvA) axonal and retrograde transduction was assayed using immunohistochemistry followed by imaging the entire brain for the presence of EGFP expression.

Electrophysiology

Mice were deeply anaesthetized, and intracardially perfused with ice-cold dissection buffer (in mM: 2.5 KCl, 1.25 NaH₂PO₄, 20 HEPES, 10 MgSO₄•7H₂O, 0.5 CaCl₂•2H₂O, 92 choline chloride, 25 glucose, 2 thiourea, 5 sodium ascorbate, 3 sodium pyruvate, and 20 NaHCO₃) bubbled with 95% O₂, 5% CO₂. Brains were then rapidly removed and immersed in ice-cold dissection buffer. DMH sections were dissected and 300 μm thick coronal slices were prepared using a vibrating microtome (Campden 7000smz 2). Slices recovered for 10 min in a 35 °C submersion chamber filled with oxygenated dissection buffer. Slices were then transferred to a secondary 35 °C submersion chamber filled with oxygenated artificial cerebrospinal fluid (ACSF; in mM: 125 NaCl, 2.5 KCl, 25 NaHCO₃, 2 CaCl₂, 1 MgCl₂, 1.25 NaH₂PO₄, 25 glucose) and allowed to recover for an additional 15 min. Slices were then kept at room temperature in oxygenated ACSF for ≥ 30 min until use (Extended Data Fig. 2b–m).

Channelrhodopsin-2 assisted circuit mapping

To isolate optically evoked inhibitory post-synaptic currents (oIPSCs) and excitatory post-synaptic currents (oEPSCs), slices were placed in a submersion chamber and perfused at 4 ml min⁻¹ with oxygenated ACSF. Cells were visualized with a Scientifica SliceScope Pro 1000 microscope equipped with infrared differential interference contrast optics. DMH^PDYN GABAergic neurons were identified by GFP fluorescence ventral to the DMC (dorsomedial hypothalamic nucleus compacta). Open-tip resistances for patch pipettes were between 2–4 MΩ and were backfilled with a Cs-based internal solution containing (in mM): 135 CsMeSO₃, 10 HEPES, 1 EGTA, 3.3 QX-314 (Cl⁻ salt), 4 Mg₂-ATP, 0.3 Na₂-GTP, and 8 Na₂-phosphocreatine with pH adjusted to 7.3 with CsOH and osmolarity adjusted to about 295 mOsM by the addition of sucrose. oEPSCs were isolated with membrane potential clamped at V_h = −70 mV and oIPSCs were isolated with membrane potential clamped at V_h = 0 mV. Bath solutions for pharmacological isolation of excitatory or inhibitory currents in whole-cell voltage clamp recordings contained SR95531 (10 μM, gabazine), kynurenic acid (1 mM), tetrodotoxin (TTX, 1 μM), and 4-Aminopyridine (4-AP, 500 μM). To photostimulate ChR2-positive fibres, an LED light source was used (470 nM, Cool LED pE-100). The blue light was focused onto the back aperture of the microscope objective (40×) producing wide-field exposure around the recorded cell of 10–15 mW per mm² as measured using an optical power meter (PM100D, Thorlabs). A programmable pulse stimulator, Master-8 (A.M.P.I.) and pClamp 10.2 and 10.6 software (Molecular Devices, Axon Instruments) controlled the photostimulation output. The oIPSC/oEPSC detection protocol consisted of one blue-light pulse (5 ms pulse length) at 30 s intervals for at least 6 consecutive sweeps. Changes in series and input resistance were monitored throughout the experiment by giving a test pulse every 30 s and measuring the amplitude of the capacitive current. Cells were discarded if series resistance rose above 25 MΩ (Extended Data Fig. 2b–m). All electrophysiology data was analysed using Clampfit 10.2 and 10.6.

Optogenetic behavioural experiments

In vivo photostimulation of LH^VGLUT2→DMH terminals was conducted by firmly attaching a fibre optic cable (1.25 m long, 200 μm core diameter, 0.63 NA; Doric Lenses) with ceramic split sleeves (Precision Fibre Products) (Fig. 2, Extended Data Fig. 3a–d). Mice were acclimated by connecting them to a ‘dummy’ fibre optic cable three days before the initiation of the experiment. Mice were stimulated with blue light (465 nM LED; Plexon) at 20 Hz, 5 ms pulses for 1 s with a 3 s recovery period (LED off) during stimulation trains to avoid ChR2 desensitization, neuronal transmitter depletion, and tissue heating. Light pulse trains were programmed using a waveform generator (National Instruments) that provided TTL input to the blue light LED. The light power exiting the fibre optic cable measured by an optical power meter (Thorlabs) was 7–8 mW in all experiments. After completion of photostimulation experiments, mice were perfused for assessment of surgical accuracy of both ChR2-expression and optic fibre tip location via histological analysis as described in ‘Stereotaxic surgeries and viral injections’.

Food intake studies

To test the sufficiency of LH^VGLUT2→DMH neurons for satiety, mice were tested under conditions of physiological hunger at the onset of the dark cycle (Fig. 2b). For dark cycle feeding, mice with ad libitum access to food were photostimulated for 5 min before the onset of the dark cycle (a time when mice often eat) and photostimulation continued throughout the duration of the study (three hours). For post-fast refeeding assays (Extended Data Figs. 1, 5), mice were food restricted for 24 h then, were given ad libitum access to food. Food was then weighed each hour to determine the amount consumed during the experimental manipulation.

Real-time place preference assays

Mice were placed in a custom-made behavioural arena (transparent acrylic, 50 × 50 × 25 cm) for 20 min (Fig. 2, Extended Data Fig. 3a–d). One counterbalanced side of the arena was designated as the photostimulation side. The mouse was placed in the stimulation side at the onset of the experiment and each time the mouse crossed to the non-stimulation side of the arena, the photostimulation immediately stopped until the animal crossed back into the stimulation side. Behavioural data was recorded with Ethovision software (Noldus Information Technologies). To test photostimulation preference during different hunger states, ad libitum-fed mice were placed within the arena immediately before the onset of the dark cycle. Following a one-week rest period, ad libitum fed mice were then placed in the same arena and tested for photostimulation preference at the onset of the light cycle. Following a one-week rest period, mice were then fasted overnight then, placed in the arena and tested for photostimulation preference at the onset of the light cycle. Each RTPP assay was counterbalanced within animal and within day.

In vivo fibre photometry

Fibre photometry was performed on a rig constructed as follows: A 465-nm LED (PlexBright LED, Plexon) was used as the excitation source which was passed through a fluorescence mini cube (excitation: 460–490 nm, detection: 500–550 nm; Doric Lenses), and transmitted onto the sample via a fibre optic cable (1 m long, 400 μm diameter, 0.48 NA; Doric Lenses). The optic fibre was coupled to the implanted optic fibre with a ceramic mating sleeve (Precision Fibre Products). Light intensity was measured as 100–200 μW at the end of the patch cord and was kept constant across sessions for each mouse. Emitted light was collected by a photodetector (2151; Newport). The signal was digitized at 1 kHz with a data acquisition card (National Instruments) and collected with a custom MATLAB (MATLAB2016a; MathWorks) script (Figs. 1–3, Extended Data Figs. 1, 3, 4).

For LH^VGLUT2→DMH axon fibre photometry recordings (Fig. 2, Extended Data Fig. 3h–j), mice underwent a 25-min recording session within their home cage that consisted of: eight ‘small’ chow (15 mg; Bio-Serv) trials, two ‘large’ chow (500 mg; Bio-Serv) trials, and one peanut butter (Reese’s peanut butter chips; Hershey) trial which were dropped into a Pyrex Petri dish. Each mouse underwent only one session per day and was food restricted (85% of ad libitum body weight) for at least one week before beginning the experiment in which it was habituated to the pellet drops within the Petri dish. All trials were pooled to calculate mean peak response (0–5 s following food presentation) to each food presentation. For object drop experiments, mice underwent a 12-min recording session within their home cage that consisted of 10 total trials of non-edible object drops. Objects consisted of uniform, white plastic marbles (BC Percision, Hungry Hungry Hippos Marbles). After an object drop session, a 500 mg chow pellet was dropped as a positive control. Each mouse underwent a single session per day. All trials were pooled to calculate mean z-score response to each object drop.

For water presentation experiments (Extended Data Fig. 3k–m), mice were water restricted (85% of original body weight) for at least one week before beginning the experiment. Mice were then habituated to receiving water in a ceramic dish within their home cage and were given free access for 5 min. All sessions were pooled to calculate the mean peak response (0–30 s) and the time to peak response.

Data were analysed using a custom MATLAB (MATLAB2016b; MathWorks) script (Figs. 1–3, Extended Data Figs. 1, 3, 4). Fluorescence traces were down-sampled from 1 kHz to 100 Hz and smoothed using a 1-s running average. The fractional change in fluorescence was calculated as ΔF/F = (F − F₀)/F₀, in which F₀ was the mean of all data points from the baseline before each trial. In home cage pellet drop or water presentation experiments, F₀ was the average for 5 s before the food drop/water presentation. In operant chamber silencing experiments, F₀ was the average for 1 s before cue presentation. All trials in a single session were averaged then the mean peak amplitude was taken for quantification.

Two-alternative forced-choice task

To determine if LH^VGLUT2→DMH neurons and DMH^LEPR neurons are necessary for food cue responses (Figs. 1–3, Extended Data Figs. 1, 4), mice underwent training in a three-nosepoke operant chamber (Bpod r2; Sanworks) controlled by a custom MATLAB (MATLAB2016a; Mathworks) script. In brief, food restricted mice (85% of ad libitum body weight) were trained to associate a light presentation with Ensure (10 μl; Ensure PLUS, vanilla) and were required to nose poke and hold their snouts within the port for 200 ms before Ensure was delivered. Light delivery was randomized between the left and right nose poke and, mice had a 10 s response window with a 10 s inter-trial interval. The required learning criterion was a success rate of ≥ 80% across three consecutive days. After mice learned the task, mice were then attached to a fibre optic cable as described above. To determine the necessity of LH afferents on food-cue responses (Fig. 3, Extended Data Fig. 4), mice underwent two, 11-min recording sessions on the same day separated by a 10-min ‘break’ period. The first session was always a saline run and mice were injected with saline 10 min before the onset of the recording session. The second session was a clozapine-n-oxide (CNO; 1 mg kg^-1; 0.5% body weight volume) or saline injection 10 min before the onset of each recording session. Comparisons between vehicle and CNO recordings were made within day; therefore, a mouse received two saline injections or, one saline and one CNO injection in a single recording session. A TTL pulse triggered at the onset of each trial determined cue onset. All trials were pooled to calculate the mean peak response (0–2 s following cue presentation) and were normalized to the first recording session within the same day.

Intragastric catheter surgery

Mice with DMH^LEPR photometry neural activity signal larger than 10% ∆F/F to chow during fast re-feed were implanted with intragastric catheters³⁸ (Extended Data Fig. 1). During surgery, mice were anaesthetized with isoflurane (1.5–3%) and treated post-operation with buprenorphine (1 mg kg⁻¹ subcutaneously) analgesia. A midline incision on the abdomen was made through skin and muscle layers. Micro-Renathane catheter tubing 6–7cm in length (Braintree Scientific, MRE-033, 0.033 × 0.014 in) was anchored with epoxy spheres on each end (Devcon Clear Epoxy Adhesive, 92926, Lowes). The catheter was inserted into the fundus of the stomach through a puncture hole and secured with surgical mesh (5-mm diameter piece, Bard, 0112660). The other end of the catheter was directed out of an intrascapular incision. A metal cap made out of 27G blunt needle was placed in the exposed end for seal. The gastric catheter was flushed with sterile water immediately, and daily, after surgery to prevent blockage. Mice were fed with gel chow diet and given at least 1–1.5 week for recovery before experimentation. Daily body weight was monitored until stable pre-surgical weight was regained.

Gastric infusion

Upon recovery, gastric infusions of liquid substances listed below were performed in a counterbalanced experimental design, under both overnight fasted and sated conditions (Extended Data Fig. 1). The intrascapularly exposed end of the gastric catheters were connected to tubing and a syringe driven by an infusion pump (Harvard Apparatus, 70-3007). At a rate of 0.1 ml min⁻¹, 1-ml infusions were performed³⁸ over the course of 10 min. Fibre photometry recordings were collected via a lock-in amplifier (TDT) and the software Synapse (TDT). Each trial was approximately 27 min (>7 min baseline recording, followed by a 10 min gastric infusion and a 10 min chow refeed). Each mouse underwent infusions of the following infusates: 0.9% isotonic saline, Ensure Original Nutrition Shake (vanilla), 1% saccharine, 25% d-glucose (equal caloric content as Ensure).

Each photometry recording data point was normalized against the average of the last 5 min of baseline period to produce the normalized traces. For normalized and delta (ΔF_n/F_n, %) quantified comparisons, we use 1-min averages at the end of baseline period (t = −1 to 0 min), 5 min into infusion (t = 4 to 5 min), and 10 min into infusion (t = 9 to 10 min). Owing to the transient nature of neural response to food, comparison of delta (ΔF_n/F_n, %) maximum magnitude between ensure infusion and food presentation were made using 30 s averages at the end of ensure infusion (t = 9.5–10 min) and at the beginning of food presentation after saline infusion (t = 10–10.5 min).

TeNT-mediated silencing

AAV-DIO-TeNT was injected into the DMH of Lepr-IRES-Cre mice, Pdyn-IRES-Cre mice, or wild-type mice (Fig. 4, Extended Data Fig. 5). Littermate controls were used for AAV-DIO-TeNT and AAV-DIO-GFP behavioural groups. Following three weeks, mice were then placed within the Bpod arena and trained everyday as described above. Mice were either food or water restricted and maintained at ≥85% ad libitum body weight. Food and water restriction was performed in separate cohorts of mice. For food-learning assays, mice were trained for a total of 21 days. For water-learning assays, mice were trained for a total of 14 days. Mice were excluded if they were non-learners meaning that they did not increase their performance rate for five consecutive days (one GFP-expressing mouse (in water-deprived group) and one TeNT-expressing mouse (in food-deprived group) were removed based on this criterion). Mice were placed in the Bpod for a total of 20 min and allowed to perform as many trials as possible with an intertrial interval of 5 s and a response window of 10 s. Mice were given either 10 μl of Ensure or 5 μl of water for food and water-learning assays, respectively. Each day, performance in the task was quantified with a custom MATLAB script (Fig. 4, Extended Data Fig. 5). It should be noted that Pdyn-IRES-Cre and wild-type behavioural cohorts (Extended Data Fig. 5–u) were different strains than our Lepr-IRES-Cre cohort; this was owing to the limited availability of mice during the COVID-19 pandemic. As such, they performed slightly differently on the 2AFC task. Therefore, their learning criterion was lowered to >70% correct responses across three consecutive days.

Quantification and statistical analysis

Statistical analyses were performed using Prism 5 and Prism 8 (GraphPad) software and are described in the figure legends in all cases. No statistical method was used to predetermine sample size, nor were randomization and blinding methods used. Statistical significance was defined as P < 0.05. All data presented met the assumptions of the statistical test employed. As mentioned in sections above, experimental mice were excluded if histological validation revealed poor or absent reporter expression or poor fibre optic placement in the region of interest. These criteria were established before data collection. n values reflect the final number of validated mice per group.

Reporting summary

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