Animals

Animals used in this study were treated in strict accordance with the national and regional ethical guidelines. All experiments were performed by FELASA-certified personnel and approved by the Animal Use and Care Committee of the Institute of Basic Medical Sciences, The Faculty of Medicine, University of Oslo, and by the Norwegian Animal Research Authority (FOTS6292, FOTS6505, FOTS6590, FOTS6720, FOTS6758 and FOTS8243). The generation of the Hcar1 knockout line and the mRFP-HCAR1 reporter mouse line has been described19. Specifically, the open reading frame of the mouse Hcar1 gene carried by the 197 kb large BAC RP23-91D24 (CHORI) was replaced by a cassette containing the mRFP, as described previously41. Both the knockout line and the mRFP line were maintained in C57Bl/6N background in Bad Neuheim and in Oslo. Genotypes were verified by Southern blotting in the mice to be used in experiments (Supplementary Fig. 7).

Animal treatment

All mice were 7–9 weeks of age at the start of the experiments. Hcar1 knockout or wild-type mice (both sexes with about equal M/F distribution, see Supplementary Fig. 1) were randomized into three groups: treadmill running, sodium L-lactate injections or saline injections (control). The mice that were treated with lactate received a subcutaneous injection of sodium L-lactate (≥99.0%, Aldrich, 71718; 2 g kg−1 bodyweight; 200 mg ml−1 dissolved in 0.9% saline; pH-adjusted to 7.4; that is, 18 mmol kg−1). The control mice received the same volume (per kg bodyweight) of 0.9% saline. The lactate or saline injections were administered subcutaneously 5 days a week for 7 weeks. The mice were weighed every week for adjustment of the dose.

Since lactate injections have been associated with acute anxiety attacks in susceptible individuals21, the mice were tested at 15 min after the first lactate injection (Supplementary Fig. 3) by the elevated zero maze, an established rodent test of anxiety42. The mice were placed on a circular white track, diameter 60 cm, width 5 cm, elevated 60 cm above the floor, with two quarters being open and two quarters closed by walls. The mouse was placed with the head facing a closed zone and allowed to explore the maze for 5 min. Their movements were recorded with a tracking camera connected to a computer with the ANY-maze Video Tracking Software (Stoelting Co., USA). The % of time spent in the closed zone and the frequency of head-dips in the open zone were recorded as positive and negative correlates of anxiety, respectively. Between the individual mice, the track was carefully wiped off (with a cloth moistened with 70% ethanol) and dried to avoid interference from the smell of other mice43.

The interval exercise regime has been previously described20,44. It is designed for optimum gain in cardiovascular function and to reach ∼90% of VO 2max during the high-intensity intervals. Briefly, each session consisted of 10 min warm-up at 5 m min−1, followed by 10 high-intensity intervals of 4 min each, and separated by 2 min of active rest. Running took place on a treadmill (Columbus Instruments, USA) at a 25 degrees incline. The mice were exposed to the high-intensity interval exercise protocol for 5 consecutive days each week, for a total duration of 7 weeks. On the first day of the exercise intervention, and then every other week (Supplementary Fig. 1), a maximal exercise-capacity test was performed for each individual mouse, to adjust the running speed of the training intervals to near the maximum they could sustain during 10 consecutive intervals. After a 15 min warm-up period at 9.6 m min−1, the band speed was increased by 1.8 m min−1 every 2 min until exhaustion, that is, the mice refused to run further, despite being manually placed back on to the band or receiving electrical stimuli (maximally 1–2 per day by the intrinsic device of the treadmill). During the 7 weeks, the maximal exercise-capacity test results, measured as maximum running speed (highly correlated with VO 2max ), levelled off at a 50% increase in capacity (Supplementary Fig. 1), showing that the training sessions were of intended intensity45. This exercise regime has been validated extensively46. Blood lactate levels at ∼10 mmol l−1 have been reported in mice during treadmill exercise at close to VO 2max (Desai and Bernstein47, Fig. 4) and at close to maximum speed19.

Plasma lactate measurements

For plasma lactate measurements, the mice were anaesthetized mildly with isoflurane and decapitated at exactly 5, 15, 30, 60 or 180 min after the dose of lactate (n=5 for each time point) or saline (n=3 for each time point). The blood was collected into 0.5 ml Minicollect tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) containing 2.5 mg sodium fluoride (NaF) and 2.0 mg potassium oxalate (KOx) per ml to prevent post vivo glycolysis (with the formation of lactate) and coagulation, respectively48. Blood samples were centrifuged, and the resulting plasma samples were frozen in liquid nitrogen and stored at −80 °C. The lactate measurements were performed using VITROS DT60II and VITROS LAC DT slides (Ortho Clinical Diagnostics, UK). The plasma samples were diluted 1:1 with water to ensure that all samples were below the maximum detection limit of the method (15 mM). All samples were analysed in duplicates.

RNA extraction and qPCR

Meninges, hippocampi, skeletal muscle (triceps surae), adipose tissue, liver and pancreas were quickly dissected out from wild-type and Hcar1 knockout mice (n=5 each, except for fat, liver and pancreas n=4; meninges from five mice were pooled in one sample for wild-type and one for knockout) and snap-frozen in liquid nitrogen and stored at −80 °C until RNA extraction. The tissue was homogenized in Qiasol lysis buffer and lysing Matrix D tubes in a Fastprep machine, at speed 6.5 for 40 s (repeated for muscle tissue). RNA was extracted using the RNeasy Lipid Tissue Mini Kit (Qiagen), according to the manufacturer’s protocol, and eluted in 30 μl RNase-free water. The Turbo DNAse (Ambion) kit was used for DNAse treatment of the extracted RNA, and the concentration and quality was measured using Nanodrop. Total RNA (2 μg) was used per cDNA reaction for all tissues except meninges (lower concentration, 1.1 μg total RNA for knockout and 1.3 μg total RNA for wild type) in a total volume of 20 μl, using the High Capacity RNA-to-cDNA Kit (Applied Biosystems by Thermo Fisher Scientific, 50 reactions kit). For qPCR, the cDNA was diluted to 5 ng μl−1, and 10 ng used per well. Power SYBR Green PCR MasterMix (Applied Biosystems, 5 ml) was used according to the protocol, with TATA-box-binding protein as a reference gene. A StepOne machine was used to run the qPCR reactions using the 2 h standard programme. To verify primer specificity, the resulting qPCR products were run on a 2% agarose gel. In addition, to verify no genomic DNA contamination, melting curve analysis was performed and negative controls were included (water and without reverse transcriptase for each sample). TaqMan-based detection was used to examine the gene expression of HCAR1 in meninges relative to the expression in the hippocampus (Supplementary Table 1, experiment 2). An amount of 200 ng total RNA was used per cDNA reaction for hippocampus and meninges in a total volume of 10 μl, using Reverse Transcriptase Core kit RT-RTCK-05 (Eurogentec, Liège, Belgium). The cDNA was diluted to 5.7 ng μl−1. Each qPCR reaction was prepared with Takyon Low ROX Probe Mastermix dTTP Blue UF-LPMT-B0701 (Eurogentec) according to the protocol, with 10 μl of diluted cDNA as template. An Agilent Mx3005P (Stratagene, La Jolla, CA, USA) machine was used to run the qPCR reactions. Ribosomal protein L27a (Rpl27a) was used as reference gene. The TaqMan probes used were Gpr81 Mm00558586_s1 and Rpl27a Mm00849851_s1, TaqMan Gene Expression Assays (Applied Biosystems by Thermo Fisher Scientific).

Quantitative western blotting

At 6 h after the end of the exercise, or 6 h after the last dose of L-lactate or saline, the mice were deeply anaesthetized with isoflurane and killed by decapitation. The hippocampi were carefully dissected out on ice and snap-frozen in liquid nitrogen. Material from in vitro experiments was processed similarly. For western blot analysis, lysates were prepared by homogenizing in radio-immunoprecipitation assay buffer (Sigma-Aldrich) containing cOmplete Protease Inhibitor Cocktail (Roche). Samples, 10 μg of total protein, were subjected to gel electrophoresis (12% SDS/PAGE (Bio-Rad)), transferred to a nitrocellulose membrane, using Transblot Turbo (Bio-Rad) and incubated overnight with the following primary antibodies: rabbit anti-VEGFA (ab46154; Abcam; diluted 1:1,000), mouse anti-α-tubulin (3873S; Cell Signaling Technologies (CST), Beverly, MA, USA; diluted 1:1,000), mouse anti-ERK1/2 (anti-p44/42 MAPK, 4696S; CST; diluted 1:1,000), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204; anti-phospho-p44/42 MAPK, 4370; CST, diluted 1:1,000), mouse anti-Akt (pan; 4691P; CST; diluted 1:1,000), rabbit anti-phospho-Act (Ser473; 4060S; CST, diluted 1:1,000). Protein bands were visualized using horseradish peroxidase (HRP)-coupled species-specific secondary antibodies (GE Healthcare Life Sciences, Oslo, Norway), that is, Amersham enhanced chemiluminescence (ECL) anti-rabbit HRP-linked whole antibody from donkey (NA934; diluted 1:20,000) and Amersham ECL anti-mouse HRP-linked whole antibody from sheep (NA931; diluted 1:50,000), combined with a chemiluminescent detection system (SuperSignal West Dura Extended Duration, Thermo Scientific, Rockford, USA). After probing the blots with phospho-specific antibodies, the blots were stripped and reprobed with antibodies to total ERK1/2 and total Akt. All antibodies produced bands corresponding to the published molecular mass of the antigen, suggesting that the antibodies were specific. Quantification of the band density was performed using the Image Studio Lite (Luke Miller) software, according to the published method (http://lukemiller.org/index.php/2013/02/analyzing-western-blots-with-image-studio-lite/).

Immunohistochemistry and fluorescence microscopy

At 6 h after the end of the exercise or 6 h after the last dose of lactate/saline, Hcar1 knockout and wild-type mice were deeply anaesthetized with zolazepam 3.3 mg, tiletamine 3.3 mg, xylazine 0.5 mg, fentanyl 2.6 μg ml−1; 0.1 ml 10 g−1 bodyweight, intraperitoneally (i.p.) and transcardially perfused with 4% paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.4 (NaPi) for 8 min. Non-treated Hcar1 knockout and wild-type mice and mRFP-HCAR1 reporter mice were anaesthetized and perfused in a similar manner. Brains from some mRFP reporter mice were viewed without perfusion or further processing (Fig. 2a,b). After perfusion fixation, organs were gently removed, viewed with a stereomicroscope (for example, Fig. 2a,b), and stored in fixative at 4 °C until cutting 50-μm-thick vibratome sections or 20-μm-thick frozen sections (after cryoprotection by immersion in 30% sucrose in 0.1 M NaPi solution overnight). Free floating brain sections were washed three times in PBS(10 mM NaPi, 0.9% NaCl) and then subjected to fluorescence immunocytochemistry, directly, or after being incubated for 30 min in citrate buffer (0.01 M, pH 8.7) at 80 °C for antigen retrieval. The sections were rinsed in PBS and unspecific binding sites were blocked by incubating the sections with 10% newborn calf serum and 0.5% Triton X-100 in PBS for 4 h. The sections were then incubated with primary antibodies (shielded from light, on an orbital shaker, overnight): mRFP signal was enhanced by rat anti-mRFP (IgG2a monoclonal, ChromoTek GmbH, Germany, code 5F8; diluted 1:500), followed by Cy3 donkey anti-rat IgG (H+L) polyclonal affinity-purified (Jackson ImmunoResearch Laboratories Inc., USA; code 712-165-150; diluted 1:500). Endothelial cells were labelled using FITC rat anti-mouse CD31 (BD Biosciences; diluted 1:200). Vascular basement laminae were labelled by rabbit anti-collagen IV (Abcam; ab6586; diluted 1:500). Fibroblast-like cells were labelled with mouse anti-vimentin monoclonal IgG 1 (Santa Cruz Biotechnology; code E-5, sc-373717; 1:250). Pericyte-like cells were labelled with rabbit monoclonal anti-PDGFRβ (Abcam; ab32570, Y92; diluted 1:100). Antibodies raised in mouse or rabbit were followed by rinsing in PBS and incubation with Alexa Fluor 488 donkey anti-mouse IgG (H+L; code A21202; Molecular Probes/Thermo Fisher, Waltham, MA) or Alexa Fluor 488 donkey anti-rabbit IgG (H+L; code A21206), both diluted 1:400 overnight. In some experiments, DAPI (Molecular Probes, Eugene, OR) was used to counterstain cellular nuclei. The sections were rinsed and mounted with Prolong Gold Antifade reagent with DAPI (Life Technologies) and cover slipped (Assistant, Germany). For the triceps surae muscle, 14 μm transverse cryostat sections were cut and mounted on glass slides. The sections were labelled as described above, but double labelling of collagen IV and chicken anti-MCT1 (diluted 1:1,000) was performed. Secondary antibodies were Alexa Fluor 488 donkey anti-chicken IgG (H+L) and Alexa Fluor 555 donkey anti-rabbit IgG (H+L), both diluted 1:400.

Surface view images were taken by an epifluorescence dissecting microscope (SteREO Lumar.V12, Zeiss, Germany, or Leica MZ6, Leica, Germany). Images of sections were acquired using confocal laser scanning microscopes (Leica TCS SP5, Leica, or LSM 6 Pascal or LSM 510 Meta, Zeiss). For quantification of capillaries in the sensorimotor cortex (Fig. 1a,b) and cerebellar cortex (Supplementary Fig. 4) fluorescence images were acquired with an Axio Scan Z1 (Carl Zeiss), imaging whole-sagittal brain sections at a resolution of 0.11 μm per pixel (Norbrain Slidescanning Facility, http://www.med.uio.no/imb/english/research/about/infrastructure/norbrain/slidescanning/).

Capillary density and diameter in the brain

Parasagittal brain sections were labelled for collagen IV as described. For hippocampus, two confocal z-stacks, 5.48 μm thick, were obtained from two separate areas in each animal, covering the whole hilus. For sensorimotor cortex and cerebellar cortex, high-resolution fluorescence images of 20 μm sections were acquired, by means of an automated slide scanner system (Axio Scan Z1, Carl Zeiss Microscopy, Munich, Germany) in order to efficiently sample large areas of brain tissue. Images were inspected using the Zen Lite Blue software (Carl Zeiss Microscopy). The quantifications were performed by an observer who was blinded with regard to treatments and genotypes. Using the SimpleGrid plug-in for ImageJ, an array of points was overlaid on the image, the hilus area of the dentate gyrus was outlined (defined as bordering on the granule cells, an on a straight line connecting the two ‘extremes’ of the dentate granule cell layer), and the number of points over capillaries (within their outer borders) were counted and compared to the total number of points over hilus (points over larger vessels and artefacts subtracted) to calculate the fraction of area occupied by capillaries. Similarly, in the cerebral cortex, the whole-cortical thickness between pia mater and white matter was sampled from 2 mm anterior to 2 mm posterior of bregma, and in the cerebellum, all three cortical layers from the entire folia 1–2 in lobus anterior were sampled. According to the Delesse Principle, the ratio of the number of points that hit the sectional profiles of the object (capillaries), to the total number of points probed on the sections through a struucture equals the ratio of the volume of the object to the volume of the entire structure49. The average capillary diameter and the section thickness affect the absolute values obtained, but not the relative changes between experiment and control (see Supplementary Table 2). Capillaries to be included in the analysis were defined as vessels no more than 10 μm in diameter24. In each mouse, the external diameter was measured in at least 10 capillaries with a visible lumen, using the ImageJ software, and averaged. The data are presented as mean±s.e.m. of four to seven mice per group, as specified.

Capillary density and fibre diameter in muscle

Transverse sections of triceps surae muscle, labelled for collagen IV (capillary) and MCT1 (muscle fibre) as described above, were examined for total area of capillaries per total area of muscle fibres, and for area of individual muscle fibres (about 30 in each condition) using ImageJ. The data are presented as mean±s.e.m. of five to six mice in each group.

Fluorescent dye angiography

To visualize brain vessels (Fig. 2e), jugular veins were retrogradely infused in situ following fixation using 1% low melting agarose (Sigma) with 50 mg ml−1 fluorescein-labelled 500 kDa dextran (Molecular Probes).

Two-photon imaging

In order to perform two-photon measurements in vivo, a cranial window was implanted over the barrel field of the somatosensory cortex in two of the mRFP-HCAR1-expressing mice, both of which showed the described cellular expression. During imaging, the mice were head-fixed and freely walking on a spherical treadmill50. We imaged the mRFP-HCAR1-expressing cells in the pia mater using a two-photon laser scanning microscope (Prairie Ultima IV, Bruker Corporation, USA). The fluorophore was excited by a Ti:sapphire laser (InSight, Spectra-Physics, USA), which was set to emit laser pulses at a wavelength of 1,000 nm, and the laser pulses were focused on the sample through a water immersion objective (N16XLWD-PF, numerical aperture (NA) 0.8, Nikon, Japan). The fluorescent signal was then filtered through a bandpass filter with a 50 nm bandwidth ∼525 nm (ET525/50 m, Chroma, USA) and captured in a photomultiplier tube. Images were acquired in the PrairieView software (Bruker Corporation).

HCAR1 immunocytochemistry

Mice (wild-type, Hcar1 knockout and mRFP-HCAR1 reporter) were anaesthetized and transcardially perfused with 4% paraformaldehyde in PBS before brain removal and post fixation in the same fixative for 24 h (ref. 51). Following dehydration in ethanol and embedding in paraffin, 4 μm thick sagittal sections through the entire forebrain were cut with a microtome (Thermo Scientific). Test sections were deparaffinized in Neoclear (Millipore) followed by rehydration through an ethanol gradient (100%, 3 min; 100%, 3 min; 96%; 1 min and 70% 1 min) and subsequently incubated at 100 °C, under pressure of 0.8−1 bar in citrate antigen retrieval buffer (pH 6.0), containing 0.05% Tween 20 (Sigma) for 2 min. Test sections were then washed in Milli-Q water and PBS containing 0.1% Tween 20. Additional sagittal brain sections derived from Hcar1 knockout mice were prepared as described above and used to pre-absorb the primary antibody solution, containing anti-HCAR1 antibody (SAB1300090 SIGMA anti-mouse Gpr81-s296 antibody produced in rabbit, affinity-isolated; 1:200 dilution), 10% goat serum, 10% BSA and 0.1% Tween 20 for 1 h at room temperature. The pre-absorbed primary antibody solution was subsequently transferred on test wild-type and Hcar1 knockout sections, mounted on microscopic slides (Thermo scientific), and incubated overnight in a humid atmosphere at 4 °C. After three washes on shaker in PBS containing 0.1% Tween 20 (5 min each), staining with Alexa Fluor 555 secondary antibody (Invitrogen) was performed in a solution containing 0.5% BSA, 0.5% goat serum and 0.1% Tween 20 for 90 min at 37 °C. After three washes in 0.1% Tween 20 in PBS (5 min each) on a shaker, samples were mounted in Wectashield mounting medium containing DAPI (Vector Laboratories). Microscopy was carried out using a Leica SP8 three-dimensional confocal microscope equipped with a × 20 water immersion lens (NA 0.75).

Hippocampal slices

Hippocampal slices were prepared from Hcar1 knockout and wild-type mice and incubated in Krebs buffer as described before18, with additions of L-lactate or the selective HCAR1 agonist 3,5-dihydroxybenzoate52, applied from neutral solution at the concentrations and for the times indicated (Supplementary Fig. 11). After incubation, the slices were extracted and processed for quantitative immunoblotting as described above.

Statistics

All data are shown as mean±s.e.m. or ±s.d. as indicated. One-way analysis of variance followed by Fisher’s least significant difference post hoc test was performed for comparison of the confocal data, Dunnett’s T3 test when variance was unequal (in muscle). For statistical analysis of western blot data, where the genotypes were analysed separately, Dunnett’s T3 post hoc test was used. P<0.05 was considered statistically significant.

Data availability

The data sets generated during and/or analysed during the current study are available from the corresponding authors on reasonable request.