All experimental protocols were conducted according to U.S. National Institutes of Health guidelines for animal research and were approved by the Institutional Animal Care and Use Committee at Janelia Farm Research Campus.
Animals were housed on a 12 hour light (06:00)/dark (18:00) cycle with ad libitum
access to water and mouse chow (PicoLab Rodent Diet 20, 5053 tablet, TestDiet), unless otherwise noted. Agrp-Cre45
, Agrp-IRES-Cre19, Pomc-Cre46
mice have been described previously. In most cases, behavioural experiments were with male mice. Females were used for some SIM1 neuron silencing experiments (6/19, ). Most experiments used Agrp-Cre45
mice, except experiments in (subset), 2c–g, 6, which were performed with Agrp-IRES-Cre19
-ChR2:tdTomato was described previously22
. The rAAV-CAG
-FLEX-hM4D:2a:GFP vector was prepared by ligating hM4D and EGFP with an intervening DNA fragment for a 2a peptide from Porcine teschovirus (GSGATNFSLLKQAGDVEENPGP), which was inserted into the rAAV2-CAG
-FLEX backbone in an inverted orientation. rAAV2/1-hSyn
-hM3D:mCherry was from the UNC viral core facility. For rAAV2-Oxytocin
-ChR2:tdTomato, we used a mouse Oxytocin
promoter fragment (−600 to −1, forward: 5′-CAAGGCCAGCCTGGTCTACACAGCAGG-3′, reverse: 5′-GGCGATGGTGCTCAGTCTGAGATCCGC-3′), followed by the Promega chimeric intron and ChR2:tdTomato, which were ligated into an rAAV2 backbone. Viral vectors were produced by the University of Pennsylvania Gene Therapy Program Vector Core or the Janelia Farm Molecular Biology Core Facility.
Viral injections and fibre placement
Viral injections were performed as described previously22
(P21 – P25 for electrophysiological recordings, P40 –P50 for behavioural experiments). ARC coordinates: bregma −1.2 mm, midline +0.2 mm; dorsal surface −5.85 mm and −5.75mm. PVH coordinates: bregma −0.7 mm; midline ±0.3 mm; dorsal surface −4.5 mm and −4.3 mm. For behavioural experiments that required photostimulation, a guide cannula was inserted (ARC: 4.5 mm, 26GA; PVH: 3.5 mm, 26GA). For PVHOXT
neuronal activation, cannula placement was through a midline craniotomy. For PBN/PVH photostimulation in the same mouse, adult male Agrp-IRES-Cre19
animals were bilaterally transduced in the arcuate nucleus with rAAV2/1-CAG
-ChR2:tdTomato (600 nL). Ferrule-capped fibres (see below) were implanted over the PVH (bregma: −0.7 mm, midline: +0.3 mm; dorsal surface: −4.0 mm) and PBN (bregma: −5.8 mm, midline: +0.9 mm; dorsal surface: −2.75 mm).
Grip cement (DENTSPLY) was used to anchor the guide cannula or ferrule-capped fibres to the skull. When needed, a dummy cannula (33GA, Plastics One) was inserted to keep the fibre guide from getting clogged. Postoperative analgesia was provided (ketoprofen, 5 mg/kg). After surgery, mice were allowed 14 – 20 d for recovery and transgene expression.
Antagonists: GABAA (picrotoxin, 50 μM, Sigma), GABAB (saclofen, 50 μM, Tocris), ionotropic glutamate receptors (AP-5, 50 μM; CNQX, 10 μM; Sigma), Npy1r (PD160170, 1 μM, Tocris), Npy2r (BIEE 0246, 0.5 μM, Tocris), and Npy5r (CGP 71683, 10 μM, Tocris), voltage-gated sodium channels (tetrodotoxin, TTX, 1 μM, Sigma). Saclofen was included to prevent potential metabotropic GABAB receptor-mediated modulation of the postsynaptic neuron. Agonist: CNO (10 μM, BioMol). Pharmacological agents were bath applied with gravity perfusion.
Electrophysiology and circuit mapping
Experimental techniques were similar to those reported previously22
. Detailed conditions for circuit mapping experiments in brain slices are in Table S1
. Coronal brain slices were prepared in chilled cutting solution containing (in mM): 234 sucrose, 28 NaHCO3
, 7 dextrose, 2.5 KCl, 7 MgCl2
, 0.5 CaCl2
, 1 sodium ascorbate, 3 sodium pyruvate and 1.25 mM NaH2
, aerated with 95% O2
. Slices were transferred to artificial cerebrospinal fluid (aCSF) containing (in mM): 119 NaCl, 25 NaHCO3
, 11 D-glucose, 2.5 KCl, 1.25 MgCl2
, 2 CaCl2
and 1.25 NaH2
, aerated with 95% O2
. Slices were incubated at 34 °C (30 min) and then maintained and recorded from at room temperature (20 – 24 °C). The intracellular solution for voltage clamp recordings contained (in mM): 125 CsCl, 5 NaCl, 10 HEPES, 0.6 EGTA, 4 Mg-ATP, 0.3 Na2
GTP, 10 lidocaine N
-ethyl bromide (QX-314), pH 7.35 and 290 mOsm/L. The holding potential for voltage clamp recordings was −60 mV unless otherwise indicated. The intracellular solution for current clamp recordings contained (in mM): 125 potassium gluconate, 6.7 KCl, 10 HEPES, 1 EGTA, 4 Mg-ATP, 10 sodium phosphocreatine (pH 7.25; 290 mOsm/L), ECl
= −75 mV. In a subset of experiments potassium gluconate-based internal solution was used for voltage clamp recordings. In most intracellular recordings, internal solutions contained GDP-βS (0.5 mM, Sigma).
For brain slice photostimulation, a laser (473 nm) was used to deliver light pulses ranging from 0.1 – 1 mW at the specimen. Laser power was monitored with a photodiode for each light pulse. Light pulse duration (1 ms) was controlled by a Pockels cell (ConOptics) and a mechanical shutter (Vincent Associates). A focal spot was targeted onto the specimen with two scanning mirrors (Cambridge Technology) through 4× or 63× objectives.
For electrical stimulation, a field electrode was placed within the ARC (for measurements from ARC) or adjacent tothe third ventricle to activate the ascending fibre tract (for measurements from the PVH). Half-maximal stimulus strength was used for asynchronous release or paired-pulse ratio measurements.
Loose-seal, cell-attached recordings (seal resistance, 20–70 MΩ, aCSF internal) were made in voltage clamp with holding current maintained at zero. Most neurons fired spontaneously. For measuring ARCAGRP
influence on spontaneous firing rate, the postsynaptic neurons were recorded while ChR2-expressing axons were photostimulated in the absence of any blockers (Supplementary Fig. 3c
, Supplementary Fig. 14d
) or in the presence of Npy1r, Npy5r, and GABAB
receptor blockers (, ). hM4D-dependent neuronal silencing was tested in POMC and SIM1 neurons in the presence of glutamate and GABAA
For axon-attached recordings, an aCSF-filled recording electrode (8 – 10 MΩ) was used, and ionotropic glutamate and GABAA receptors were blocked. After a subset of recordings, TTX (1 μM) was used to confirm that signals were due to action potentials. AGRP or POMC axons in the PVH were identified and targeted by tdTomato fluorescence.
For asynchronous release measurements, CNQX, AP5 and saclofen were present. Asynchronous release was also prominent in the absence of saclofen (, ). For analysis of asynchronous release, traces from 8–10 trials were averaged. The DC component during photostimulation was calculated by measuring the current amplitude 2 ms before each photostimulus and was normalised to the peak amplitude of the first synchronous synaptic response (). Decay times for the delayed asynchronous component in the train stimulus were calculated by a single exponential fit starting 100 ms following last light pulse (). Cumulative charge from delayed release was calculated as the total area under the averaged traces 100 ms – 3 s following last pulse (). For baseline charge transfer a 3 s pre-stimulus window was used.
Quantal amplitude measurements () were performed under the same conditions as above except that, in the aCSF, 2 mM Ca2+ was replaced by 2 mM Sr2+. Quantal events were chosen from a window immediately following stimulation until the event frequency dropped to three times above the baseline spontaneous event frequency.
In vivo photostimulation
Components for food consumption monitoring and photostimulation were similar to those reported previously4
. Light was delivered to the brain through an optical fibre (200 μm diameter core; BFH48–200-Multimode, NA 0.48; Thorlabs), which was implanted through the fibre guide the day before photostimulation. The fibre tip was positioned to a distance of ~0.8 mm from the targeted region. The relationship of light scattering and absorption in the brain as a function of distance has been described previously50
. Using this relationship, the light power exiting the fibre tip (10 – 15 mW) was estimated to correspond to >2.0 mW mm 2
at the ARC or PVH. For optical delivery of light pulses with millisecond precision to multiple mice, the output from a diode laser (473 nm, Altechna) was split into eight beams using a combination of 50/50 beam splitters and turning mirrors (Thorlabs). The main output beam from the diode laser was controlled using an acousto-optic modulator (AOM) (Quanta Tech, OPTO-ELECTRONIC) to generate light pulses that were launched into separate fibre ports (PAF-X-5 or PAF-X-7, Thorlabs) and their corresponding optical fibres. Using these components, eight mice could be simultaneously photostimulated. For all in vivo
photostimulation experiments the same pulse protocol was used: 10 ms pulses, 20 pulses for 1 s, repeated every 4 s for 1 h.
For ARCAGRP→PVHOXT photostimulation experiments in which ipsilateral and contralateral sides were dissociated (), mice with missed injections, bilateral injections (either in ARC or PVH) or low Fos expression in AGRP neurons were excluded.
For bilateral OXT neuron photostimulation following food deprivation, (Supplementary Fig. 14l
) experiments were performed in mice bilaterally infected with rAAV2/1-Oxytocin
-ChR2-tdTomato that were food deprived for 24 hours. Before re-feeding (5 min), photostimulation was initiated through a cannula placed over the PVH midline. Mice were allowed ad libitum
access to food for 2 days after which OXT neurons were photostimulated again for 1 hour. Immediately following photostimulation, mice were perfused and their brains were fixed, sectioned, and stained for Oxt- and Fos-immunoreactivity (Supplementary Fig. 14i
For experiments in which ARCAGRP
→PVH and ARCAGRP
→PBN projection stimulation were in the same animal, fibres were capped with 1.25 mm OD zirconia ferrules (see http://syntheticneurobiology.org/protocols/protocoldetail/35/9
), implanted into the brain, and affixed to the skull of the animal with dental cement. For light delivery, the implanted ferrule-capped fibre was coupled to another optical fibre with a matching 1.25 mm OD zirconium ferrule using a zirconium sleeve.
Pharmacology of AGRP neuron evoked feeding
Surgeries and photostimulation were similar to ARCAGRP→PVH stimulation experiments as described above except that cannula placement over the PVH was 0.25 mm lateral to the midline with other coordinates being the same. Prior to photostimulation (−10 min), vehicle [0.15 M saline, DMSO (10%), glacial acetic acid (2.5%)], Npy1r antagonist (BIBO-3304, 3 μg, Tocris), or GABAA receptor antagonist (bicuculline methiodide, 2.5 pmol, Sigma) were delivered on separate days through the same cannula that was used for fibre implantation. Antagonist injections were on the second or third day, counterbalanced between groups. Injections (50 nL) were through an injection cannula (33 Gauge) coupled to a Hamilton syringe that was driven by a Narishige micromanipulator (~30 nL/minute). Animals were subsequently photostimulated (1 h). Two animals had cannula blockage on the final day of injection (condition: bicuculline methiodide followed by photostimulation) and were eliminated from this analysis group.
PVH pharmacology experiments with the hM3D neuronal activator were performed as above, except that, instead of light delivery, mice were injected with CNO (intraperitoneal, 0.3 mg/kg, also see below) immediately prior to intracranial antagonist injection.
In separate mice, the effective concentration of BIBO-3304 was determined by the ability to block food intake evoked by Npy (70 pmol, Sigma) injection into the PVH. Consistent with earlier reports51
, Npy-dependent food intake was suppressed. We further confirmed that this concentration does not induce a nonspecific inhibition of overall feeding response, by delivering the same dose into the PVH of 24 hour food deprived animals. While 3 μg BIBO-3304 effectively blocked Npy-induced food intake, it did not significantly decrease re-feeding after deprivation, although there was a trend for reduced food intake (Supplementary Fig. 16
For hM4D-dependent silencing experiments, rAAV2/1-CAG
-hM4D:2a:GFP virus injections were made bilaterally. Mice with total misses or unilateral injections were excluded from analysis after post hoc
examination of GFP expression. CNO (5 or 0.3 mg/kg) or saline was delivered by intraperitoneal injection. Control saline injections contained an equivalent amount of DMSO (0.6%). Consistent with a previous report5
, we found that CNO injection alone did not stimulate food intake in uninfected mice (data not shown).
For PSAML141F-GlyR silencing experiments in SIM1 neurons, we bilaterally transduced Sim1-Cre mice with rAAV-Synapsin-FLEX-rev-PSAML141FGlyR:IRES:EGFP as described above for hM4D transduction of the PVH. A cognate ligand for this chimeric chloride channel, PSEM308 (5 mg/kg) was dissolved in saline and administered intraperitoneally. Food intake was measured before (Pre) and after PSEM308 administration (1 h each). PSEM308 (5 mg/kg) was also administered to untransduced control mice.
Progressive ratio task
For the entire training protocol, all animals were maintained under ad libitum fed conditions, and were never pre-exposed to food deprivation or neuronal manipulation before the days on which these were tested. Agrp-Cre or Sim1-Cre mice were first acclimated to handling and were exposed to the testing arena in 3 sessions, where they became familiar with the pellet delivery system and food retrieval. To continue training, each animal had to consume at least 5 pellets from the food hopper on the last session. Animals were then trained to perform lever pressing for food pellets.
The response lever was placed adjacent to the food delivery cup. Animals were allowed one hour in the arena to perform Fixed Ratio (FR)1, FR3, and FR5, each for 3 days. After 5 training sessions, animals that were not pressing a lever sufficiently to earn at least 3 food pellets, were eliminated from further training and testing. To test whether lever pressing was reinforcer-directed, a second identical but inactive lever was placed in the arena on the opposite side of the food delivery cup on the third FR3 session. In successive training and experimental sessions, the inactive lever was rarely pressed as it never led to a food reinforcer.
For AGRP neuron activation experiments, break point testing was performed on a progressive ratio-2 schedule, such that each successive food reward increased the lever-press schedule by two additional responses. Break point is defined here as the lever-press schedule reached at the end of the one hour session52
. The following day, an optical fibre was inserted into the guide cannula. The animal was allowed 1 hour in its home cage to habituate after handling. After transfer to the behavioural test cage, the progressive ratio schedule was repeated in the ad libitum
fed animal with photostimulation. The following day, food was removed from the home cage (24 h) with water freely available, followed by a break point test under food deprived conditions. After allowing at least 3 days for ad libitum
repletion, the break point was tested again.
Progressive ratio experiments for SIM1 neuron silencing were performed similarly, except that each test session was extended to 2 hours due temporal variability for pharmacogenetic experiments following intraperitoneal injection. Due to the increased session length, a PR3 schedule was used to prevent satiation. Mice received saline injections in each test session except the day in which behavioural effect of SIM1 neuron silencing was tested. On that day they received a single dose of CNO (5 mg/kg, IP) immediately before being placed in the test cage. The experimenter was blind to the ChR2 or hM4D expression of the subjects, which was revealed after post hoc histology.
Image analysis of PVH-selectivity for SIM1 neuron transduction with hM4D was in Image-J. Confocal images across the rostro-caudal axis, included the PVH as well as neighbouring structures (1.3 mm × 1.3 mm), were subjected to the automated thresholding function, and total thresholded surface area was measured. Fluorescence within and outside of the PVH was calculated (n
= 6 mice). hM4D-transduced SIM1 neurons were primarily located within the PVH (84 ± 2% of total fluorescence, n
= 6 mice), and there was a negative correlation between break point and the small proportion of scattered hM4D-expressing neurons that extended outside the PVH (r
= −0.7, Supplementary Fig. 13g
anti-Agrp (1:5000, goat, Neuronomics), anti-Agrp (1:2000, rat, Neuronomics), anti-Fos (1:5000, rabbit, Santa Cruz), anti-αMSH (1:500, sheep, Millipore), anti-Pomc (1:200, rabbit, Phoenix Pharmaceuticals), anti-Oxytocin (1:3000, mouse, Abcam), anti-Synapsin I (1:1000, rabbit, Millipore), anti-tdTomato (1:20000, guinea pig, Covance), anti vGat (1:2000, rabbit, SySy). Fluorophore-conjugated secondary antibodies were from Invitrogen and Jackson Immuno. Antibodies were diluted in phosphate buffered saline, 1% BSA, 0.1% Triton X-100.
Immunohistochemistry and imaging
After mice were used for behavioural experiments, they were transcardially perfused with 4% paraformaldehyde/0.1 M phosphate buffer fixative. Tissue was post-fixed in this solution for 4 – 5 hours and washed overnight in phosphate buffered saline (pH 7.4). Brain sections (50 μm) were processed for immunohistochemistry, mounted on glass slides using VECTASHIELD mounting medium with 4′,6-diamidino-2-phenylindole (DAPI), and coverslipped for imaging.
Photostimulation experiments were confirmed by post hoc Fos quantification. After experiments were complete, mice were photostimulated (1 h) in the absence of food and immediately processed for perfusion, followed by sectioning as described above. Transduction of AGRP, SIM1 or OXT neurons was evaluated by the expression of ChR2-tdtomato. After anti-Fos immunohistochemistry, nuclei were stained with DAPI, and confocal images (2 μm thickness, 5 images) were collected using a 20× (0.8 N.A.) objective, and ChR2 neurons were counted in a single section at the centre of the stack (stacks above and below were also examined to minimise false-negative reporting of Fos expression). Only cells that clearly had a nucleus demarcated by the presence of DAPI staining and surrounded by membrane-localised ChR2-tdtomato fluorescence were included. A subset of those neurons also had Fos-immunoreactivity overlapping with DAPI, and these neurons were taken as Fos-positive ChR2 neurons. Large DAPI-positive nuclei without any surrounding tdtomato fluorescence were considered ChR2-negative neurons. For each mouse, Fos-positive counts from three sections were averaged across the rostral-caudal axis of the ARC or PVH.
To quantify axonal ChR2-penetrance for PVH/PBN stimulation experiments, brain slices were immunostained for tdTomato to enhance detection, and confocal images were collected. The percentage of AGRP-containing varicosities transduced with ChR2:tdtomato in the PVH and the PBN were calculated using automated varicosity-detection in Vaa3D53
(confirmed by manual inspection). At least 300 varicosities from three distinct sections along the rostral-caudal axis of the PVH and the PBN were analysed from each animal.
Values are represented as means ± s.e.m. P values for pair-wise comparisons were calculated by two-tailed Student’s t-test. P values for comparisons across more than two groups were adjusted with the Holm-Sidak correction. Linear regressions and tests involving 1-way and 2-way ANOVA with one factor repetition were calculated with SigmaPlot (Systat). n.s. P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.