Animal handling

Animal procedures were in accordance with the NIH guidelines and approved by the NYU School of Medicine Animal Care and Use Committee. All animals were on 12 h light:dark cycle, with lights on from 06:00 to 18:00; ex vivo slices were prepared between 08:00 and 12:00. Mechanistic studies in AL rats and mice were conducted in ex vivo slices from animals housed in pairs, whereas rats were singly housed for all diet group comparisons and for behavioural studies.

Rat diet regimens

Adult male Sprague–Dawley rats (Taconic) were 8–10 weeks old at initiation of diet regimens lasting 21–30 days. Rats were semi-randomly assigned to the diet groups: subjects were ranked by initial weight, then each successive trio of rats was distributed randomly among the diet groups. AL rats had free access to rat chow for the same period as paired rats on FR or OB diets. All rats had free access to water. Food restriction was implemented as previously51; briefly, rats received 40–50% of AL intake of standard rat chow daily until body weight was reduced by 20%, after which food was titrated to maintain this weight. OB rats had free access to rat chow and chocolate Ensure, a highly palatable liquid with moderately high fat and sugar52.

Forebrain ChAT knockout mice

Mice with a conditional floxed allele of ChAT (ChATflox) were crossed with a Nkx2.1Cre transgenic line to produce mice in which ablation of ACh synthesis is restricted to forebrain32. Non-mutant transgenic littermates were controls; their genotypes Cre+;ChATflox/+ and Cre−;ChATflox/flox are referred to as ‘heterozygotes’. Adult male mice used for slice studies had ad libitum access to chow and water.

Ex vivo slice preparation and physiological solutions

Rats or mice were deeply anaesthetized with 50 mg kg−1 pentobarbital (intraperitoneal (i.p.)) and decapitated. For voltammetry, coronal forebrain slices (300–400-μm thickness) were cut on a Leica VT1200S vibrating blade microtome (Leica Microsystems; Bannockburn, IL) in ice-cold HEPES-buffered artificial CSF (aCSF) containing (in mM): NaCl (120); NaHCO 3 (20); glucose (10); HEPES acid (6.7); KCl (5); HEPES sodium salt (3.3); CaCl 2 (2); and MgSO 4 (2), equilibrated with 95% O 2 /5% CO 2 . Slices were then maintained in this solution at room temperature for 1 h before experimentation30,32,53. For electrophysiology, after anaesthetization, rats were perfused transcardially with ice-cold solution containing (in mM): sucrose (225); KCl (2.5); CaCl 2 (0.5); MgCl 2 (7); NaHCO 3 (28); NaH 2 PO 4 (1.25); glucose (7); ascorbate (1); and pyruvate (3), and equilibrated with 95% O 2 /5% CO 2 . Slices were cut in this solution, then transferred to a recovery chamber in modified aCSF containing (in mM): NaCl (125); KCl (2.5); NaH 2 PO 4 (1.25); NaHCO 3 (25); MgCl 2 (1); CaCl 2 (2); glucose (25); ascorbate (1); pyruvate (3); and myo-inositol (4), equilibrated with 95% O 2 /5% CO 2 ; this solution was initially at 34 °C, then allowed to cool gradually to room temperature54. All voltammetry and physiology experiments were conducted in a submersion recording chamber at 32 °C that was superfused at 1.5 ml min−1 with aCSF containing (in mM): NaCl (124); KCl (3.7); NaHCO 3 (26); CaCl 2 (2.4); MgSO 4 (1.3); KH 2 PO 4 (1.3); and glucose (10), and bovine serum albumin (BSA, 0.05–0.1 mg ml−1) equilibrated with 95% O 2 /5% CO 2 ; slices were allowed to equilibrate in this environment for 30 min before experimentation.

Fast-scan cyclic voltammetry

Evoked DA release studies were conducted using FCV in brain slices32,53 prepared from male rats or ChAT forebrain knockout mice and heterozygote controls (5–8 weeks). Studies in ChAT knockout mice were blinded, but rat diet groups had obvious phenotypes that precluded blinding. Voltammetric measurements were made with a Millar Voltammeter (available by special request to Dr Julian Miller at St Bartholomew’s and the Royal London School of Medicine and Dentistry, University of London). A conventional triangle waveform was used for FCV, with a scan range of −0.7 to +1.3 V (versus Ag/AgCl), scan rate of 800 V s−1, and sampling interval of 100 ms30,32,53. Data were acquired using a DigiData 1200B A/D board controlled by Clampex 7.0 software (Molecular Devices). DA release was evoked using a concentric stimulating electrode; stimulus pulse amplitude was 0.4–0.6 mA and duration was 100 μs30,32,53. Local single-pulse stimulation was used in NAc core and CPu; however, a brief high-frequency pulse train (five pulses at 100 Hz) was used to amplify evoked [DA] o in NAc shell. Both stimulus paradigms evoke DA release that is action potential and Ca2+ dependent, unaffected by concurrently released glutamate and GABA42,55, and facilitated by concurrently released ACh29,30,31,32,33,34. To quantify evoked [DA] o , electrodes were calibrated with known concentrations of DA at 32 °C after each experiment in aCSF and in the presence of each drug used during a given experiment53.

Voltammetry experiments to assess the effect of insulin on evoked [DA] o were obtained using either of two protocols. Initial experiments to determine the time course for the effect of insulin (Sigma, I5523) were performed by monitoring evoked [DA] o every 5 min in a single site. Insulin was applied after consistent evoked [DA] o was obtained (typically 4–5 measurements); the effect of insulin was maximal after 50–60 min, and then evoked [DA] o remained at this level for the duration of the experiment (typically 90 min total insulin exposure; Fig. 1c). Subsequently, the effect of insulin was assessed by recording evoked [DA] o at 4–5 discrete sites in slices (+1.5 mm from bregma) in each of three striatal subregions under control conditions (aCSF or aCSF plus drug) and again at the time of maximal insulin effect (sampling over 60–80 min), then these samples were averaged for each subregion. The effect of insulin diminished with time after slice preparation; to minimize time ex vivo and to optimize animal use, typically, two slices from a given animal were tested in the recording chamber at the same time. Drugs used to challenge the effect of insulin were applied 15 min before insulin via superfusing aCSF, including HNMPA trisacetoxymethyl ester (HNMPA-AM 3; Enzo Life Sciences), S961 (Novo Nordisk), LY294002 (Sigma), picropodophyllotoxin (PPP; Tocris), mecamylamine (Tocris) and DHβE (Tocris). As described in Results, possible altered sensitivity of nicotinic ACh receptors among diet groups was tested by comparing the ratio of peak [DA] o evoked by 5 p (100 Hz) with that evoked by 1 p evoked (5 p:1 p ratio)30,31 in NAc core in the presence of 0–500 nM nicotine (Sigma).

Determination of V max from evoked [DA] o transients in striatal slices

To evaluate insulin-induced changes in DAT-mediated DA uptake, the initial portion of the falling phase of evoked [DA] o curves was fitted to the Michaelis–Menten equation to extract V max (maximal uptake rate constant)56. K m (which is inversely related to the affinity of the DAT for DA) was fixed at 0.2 μM and is known to be similar across striatal subregions57 and unaffected by insulin (see Supplementary Fig. 4 caption).

Whole-cell recording

Brain slices were prepared from 29- to 35-day-old male rats; recording conditions were identical to those used in DA release studies. Whole-cell current-clamp recordings used conventional methods54. Striatal ChIs were visualized using an Olympus BX51WI microscope (Olympus America, Center Valley, PA) with infrared differential-interference contrast optics and a × 40 water-immersion objective. The pipette solution contained (in mM): K-gluconate (129); KCl (11); HEPES (10); MgCl 2 (2); EGTA (1); Na 2 -ATP (2); Na 3 -GTP (0.3); and adjusted to pH 7.2–7.3 with KOH. For recorded neurons to be assessed for ChAT immunoreactivity, 0.3% biocytin was included in the pipette solution and the neurons recorded briefly (∼5 min) to minimize dilution of intracellular contents. Pipette resistance was ∼3–5 MΩ. Recordings were obtained using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale CA) and low-pass filtered at 2 kHz. ChIs were identified by established electrophysiological criteria28; most were tonically active initially, but activity diminished variably after patching. However, responsiveness to current injection was generally robust and consistent over time, and was therefore used to investigate the effect of insulin on ChI excitability (see Results). In experiments to examine the role of InsRs and IGF-1Rs in this response, either HNMPA or PPP was applied for at least 20 min before a ChI was patched. Maximal effects of insulin alone were typically observed after ∼16 min of exposure, although in some cells, the increase was not maximal until 50 min or longer. Moreover, in four of six neurons recorded in PPP, insulin caused an initial decrease in spike number before recovering to and exceeding starting spike number. Consequently, in all experiments, the effect of insulin was quantified by comparing the maximum effect on spike number with the number of spikes evoked immediately before insulin application. The apparent difference in the time to reach peak effect could reflect a number of factors, including the depth of the recorded cell in the slice. Evoked action potentials were also recorded in ChIs in the absence of insulin at comparable time points.

High-performance liquid chromatography

The DA content of rat striatal slices (400-μm thickness) was determined using high-performance liquid chromatography with electrochemical detection58. Slice pairs were equilibrated for 30 min at 32 °C in aCSF, and then one slice per pair was incubated for an additional 60 min at 32 °C in aCSF while the other was incubated in aCSF with 10 or 30 nM insulin. For diet group comparisons, striatal tissue was collected between 30–60 min post-recovery. Following incubation, excess aCSF was carefully removed from slices, a sample of striatal tissue (7–10 mg) was weighed, frozen on dry ice and then stored at then stored at −80 °C. On the day of analysis, samples were sonicated in ice-cold, eluent, deoxygenated with argon58, spun in a microcentrifuge for 2 min, and the supernatant injected directly onto the HPLC column (BAS, West Lafayette, IN); the detector was a glassy carbon electrode set at 0.7 V versus Ag/AgCl.

Immunohistochemistry

For immunohistochemical labelling, rats were anaesthetized with sodium pentobarbital (50 mg kg−1, i.p.), then perfused transcardially with PBS (154 mM NaCl in 10 mM phosphate buffer, pH 7.2) followed by 4% paraformaldehyde in this PBS; brains were removed and coronal sections (20 μm) were cut and processed conventionally27,59. Immunofluorescence images were obtained with a Nikon PM 800 confocal microscope equipped with a digital camera controlled by Spot software (Diagnostic Instruments Inc.) and using a × 100 objective (numerical aperture=1.4) or with a Zeiss LSM 510 confocal microscope using a × 63 objective (numerical aperture=1.2). The lasers were Argon (488 nm), He/Ne (543 nm) and He/Ne (633 nm). Appropriate filters for each laser were selected by the confocal microscope software. Pinhole size varied with the objective used and section thickness selected in z-stack generation; we chose the optimum pinhole value indicated by the software (typically 30 μm). Digital files were analysed with deconvolution software (AutoQuant Imaging), with final images processed using Adobe Photoshop 7.0. All images were adjusted for brightness and contrast; such adjustments were made uniformly to all parts of the image. Striatal DA axons were identified using two TH antibodies: polyclonal AB152 rabbit anti-TH (1:800) and monoclonal MAB318 mouse anti-TH (1:500) (both from Chemicon). Three InsR antibodies were used: sc-57342 and sc-09 (1:100; Santa Cruz), and PP5 (a gift from Pfizer). The specificity of each has been demonstrated previously60,61, and was confirmed in the present studies by the absence of immunolabelling with antibodies sc-57342 or PP5 in the presence of the corresponding blocking peptide. ChAT antibody was AB144 (1:200; Millipore), and biotin was from Vector (1:200). Secondary antibodies used were donkey anti-rabbit Alexa 488 (Invitrogen), or donkey anti-rabbit Cy2 (Jackson Laboratory, Bar Harbor, ME), donkey anti-goat Cy3 (Jackson) and donkey anti-mouse Cy5 (Jackson).

To evaluate co-localization of InsRs in TH+ axons, we used methods described previously to identify the presence of Kir6.2, the pore-forming subunit of ATP-sensitive K+ channels in DA axons27. Puncta representing InsRs were distributed throughout adjusted images, indicating that superimposition with TH immunoreactivity may occur to some degree by chance. To test this assumption, we counted InsR/TH superimpositions in 42 independent fields in three NAc immunolabelled sections from two rats. The InsR digital files were then rotated 90° clockwise and the counts repeated; rotation decreased the number of InsR puncta that co-localized with TH in most fields (see Results). The decrease in the number of superimpositions with rotation27 indicated the proportion of InsR puncta in each striatal field associated with DA axons.

Blood glucose and insulin ELISA

Trunk blood was collected at time of decapitation for slice studies. Blood glucose was determined immediately with a standard blood glucose monitor. For insulin, additional blood was collected in EDTA-containing tubes and centrifuged at 1,500g for 15 min; supernatant (plasma) was collected and stored at −80 °C until processing with an ALPCO Rat Insulin ELISA kit.

Cannula placement and histological verification

Forty one adult male Sprague–Dawley rats (Taconic and Charles River) initially weighing 350–425 g were anaesthetized with ketamine (100 mg kg−1, i.p.) and xylazine (10 mg kg−1, i.p.) and stereotaxically implanted with two chronically indwelling guide cannulae (26 gauge) placed bilaterally 2.0 mm dorsal to infusion sites in the NAc medial shell62 (1.6 mm anterior to bregma; 2.1 mm lateral to the sagittal suture, tips angled 8° towards the midline, 5.8 mm ventral to skull surface). Rats were given banamine (2.0 mg kg−1, subcutaneous) as a post-surgical analgesic following recovery from anaesthesia and the morning after. One week after surgery, rats were placed on FR (described above) and maintained at 80% of their post-surgical recovery weight for the remainder of the study. Cannula placement was determined histologically after completion of behavioural testing. Each rat was killed with CO 2 , decapitated and the brain removed and fixed in 10% buffered formalin for >48 h. Frozen coronal sections (40-μm thickness) were cut on a Reichert-Jung Cryostat, thaw mounted on gelatin-coated glass slides, and stained with cresyl violet. Data from a given rat were used only if both cannulae were within the medial NAc shell62 (including the shell/core or shell/olfactory tubercle border) (Supplementary Fig. 5); based on these criteria, two rats were excluded from the final analysis.

Flavour-preference conditioning pre-exposure

Rats received one overnight (in home cage) and six 30-min-per-day sessions of pre-exposure (in testing chambers) to 0.2% sodium saccharin (Sigma) in water with a 48-h interval between sessions. Rats then received two 5-min-per-day sessions of exposure to 0.2% sodium saccharin in 0.05% unsweetened grape or cherry Kool-Aid (Kraft Foods) in water. For the first Kool-Aid pre-exposure session, half of the rats received cherry-flavoured solution, and the other half received grape-flavoured solution. The flavours were reversed on the second Kool-Aid pre-exposure session to ensure that all rats sampled each flavour. Intake was measured for all pre-exposure sessions. Testing chambers were clear plastic cages with fresh bedding. For all pre-exposure sessions, rats had access to the same solution on both sides of the chamber. Except for the overnight pre-exposure session, all sessions were conducted in a behavioural procedure room, with a 30-min habituation period before any training or testing.

One-bottle conditioning

Previous studies have shown that microinjection of InsAb into ventromedial hypothalamus can block the effect of insulin on feeding behaviour and glucagon secretion63,64. Here we used this approach to assess a possible role of insulin in the reinforcement of food choice. Rats were semi-randomly assigned based on average pre-exposure intake volume into two groups, control or experimental (InsAb). In the control group, rats received vehicle (microinjection PBS; 137 mM NaCl and 2.7 mM KCl in 10 mM phosphate buffer) or IgG (Abcam ab81032; 0.5 μg μl−1 in PBS, as received) microinjection in NAc shell before consuming one of the two flavoured solutions, and a mock microinjection before consuming the other flavoured solution. In the experimental group, rats received NAc shell microinjection of InsAb (Abcam ab46707; 0.5 μl of 1 μg μl−1 in PBS, as received) before exposure to one flavoured solution, and mock microinjection prior exposure to the other. Two sets of subjects with alternation between fluid microinjection and mock microinjection were used, so that the total number of microinjections was limited to four, thereby minimizing possible tissue damage and loss of sensitivity at the microinjection site65. For fluid microinjection, control solution or InsAb was loaded into two 30-cm lengths of PE-50 tubing attached at one end to 5 μl Hamilton syringes filled with distilled water and at the other end to 31-gauge injector cannulae, which extended 2.0 mm beyond the implanted guides. The 0.5 μl infusion volumes were delivered over 90 s at a rate of 0.005 μl s−1; the injector was left in place for ∼60 s to allow time for diffusion, then the injector was replaced with the stylet.

Rats were transferred directly to the behavioural chambers within 2 min of completion of the microinjection or mock microinjection. Conditioning solutions contained 0.2% sodium saccharin, 0.05% unsweetened grape or cherry Kool-Aid, and 0.8% glucose. Solution access was limited to 30 min per session. Paired flavour and side of the chamber with drinking access were semi-randomly assigned and counterbalanced in each group. The interval between microinjections was at least 72 h, alternating between infusion and mock sessions for a total of eight conditioning sessions.

Two-bottle preference test

Forty-eight hours after the last conditioning session, rats were placed in testing chambers with simultaneous access to both conditioning flavours; solutions were 0.2% sodium saccharin in 0.05% grape or cherry Kool-Aid, without glucose. Testing occurred over 2 days (60 min per day). The position of the drinking tube containing mock-paired or infusion-paired solution was alternated to ensure that each rat was tested for consumption of each solution on both sides of the cage. Intake of each flavoured solution was averaged for the two test days to determine preference.

[3H]DA uptake in striatal synaptosomes to assess InsAb efficacy

Striatal synaptosomes21,66 were prepared from AL rats (male, 350–400 g), with NAc (shell and core) and CPu dissected and prepared separately. Tissue from each region was homogenized in 15 volumes of an ice-cold 0.32 M sucrose solution in a glass homogenizer with motor-driven Teflon pestle; after rinsing and centrifugation, the final pellet was re-suspended in ice-cold 0.32 M sucrose21,66. Before initiating the [3H]DA uptake assay66, synaptosomal aliquots in a total volume of 180 μl of uptake buffer were incubated in a shaker for 15 min at 30 °C in the presence or absence of 30 nM insulin, in vehicle (PBS) or in InsAb (final dilution 1:500), in IgG (final dilution 1:500) or in vehicle. Uptake buffer contained (in mM): NaCl (122); Na 2 HPO 4 (3); NaH 2 PO 4 (15); KCl (5); MgSO 4 (1.2); glucose (10), CaCl 2 (1); nialamide (0.01); tropolone (0.1); and ascorbic acid (0.001), pH 7.4. Uptake of [3H]DA was initiated by rapidly dispensing 20 μl of each synaptosomal suspension into the 96-well plates with varying concentrations of DA (0.003–1.0 μM) and [3H]DA (5 nM); after 5 min in a plate-shaker at 25 °C, uptake was terminated by cold, rapid vacuum filtration66. Counts per well were converted to pmoles, then corrected to mg of total protein per minute. All assays were performed in triplicate and repeated at least four times; V max and K m were calculated using Biosoft Kell Radlig software (Cambridge, UK).

Statistical analysis