AVV.PRSx8.RFP

All adenoviral vectors (AVV) were produced by standard homologous recombination51. To enable the visualization of NEergic neurons without concomitant opto-excitation, we expressed a monomeric red fluorescent protein52 under the control of the PRSx8 promoter, which is selectively active in NEergic neurons in the LC53.

AVV.sPRSx8.ChIEF-tdTomato

To optically excite NEergic neurons, we expressed a fusion (kindly provided by Dr John Y. Lin, UCSD) between ChIEF, a light-sensitive cation channel with improved responses to high frequency stimulation, and a bright red fluorescent protein tdTomato, under the control of the PRSx8 promoter.

AVV.sGFAP.ChR2(H134R)mKate

In patch clamp experiments, we used a fusion of a mutant of channelrhodopsin2, ChR2(H134R)54, with the red fluorescent protein Katushka1.3 (ref. 55). This allows excitation of astrocytes with blue light delivered by an additional laser or LED and visualization of transduced cells using green or yellow laser line of the confocal microscope. A compact transcriptionally amplified version of glial fibrillary acidic protein (GFAP) promoter was employed to enhance and target expression to astrocytes56,57. This AVV has previously been shown to elevate [Ca2+] i levels and activate astrocytic signalling and transmitter release4. Although we did not further investigate the mechanism of L-lactate production triggered by this construct, it is important to state that it is highly unlikely to be a result of membrane depolarizations characteristic of ChR2-like constructs. Rather, it is most likely a consequence of an increase in [Ca2+] i because the key regulator of glycogen breakdown, phosphorylase kinase can be directly activated by Ca2+-calmodulin, this pathway is well known and is described in various textbooks. In addition, several adenylyl cyclase isoforms that are expressed in the brain can be directly activated by a Ca2+-calmodulin58,59, for review see ref. 60. It is therefore feasible that Ca2+-calmodulin activates adenylyl-cyclase and cAMP thus formed acts by activating PKA, which phosphorylates phosphorylase kinase that in turn phosphorylates and activates glycogen phosphorylase.

AVV.sGFAP.optoβ 2 AR

To activate astrocytic G-protein-coupled receptor-mediated intracellular signalling, we employed an opsin-β 2 -adrenergic receptor chimera (optoβ 2 AR), which is derived from extracellular and transmembrane parts of rhodopsin and the intracellular domains of the β 2 -adrenergic receptor20. Excitation of optoβ 2 AR by blue light recruits the G s PCR pathway, leading to the activation of adenylate cyclase (AC) and increase in cAMP (ref. 20). The astrocyte-selective promoter sGFAP (see above) was used to express optoβ 2 AR in astrocytes specifically.

The use of optoβ 2 AR only for FCV was determined by difficulties in handling cells expressing these constructs in patch clamp experiments. While establishing whole-cell configuration, it was essential to visualize the neurons for prolonged periods of time and with fairly intensive light. Compared with ChR2, we found it difficult to avoid unsolicited activation of astrocytes when optoβ 2 AR were used, possibly due to the wider excitation spectrum of these receptors. This construct also has a very narrow window for functional expression, while its overexpression is detrimental to astrocytes. Although these vectors are based on the same expression system as we used previously, for this study we have performed additional studies to verify lack of ‘leak’ expression in LC neurons with sGFAP-based vectors. Fourteen slices from four separate preparations were studied using confocal microscopy. Of 438 identified DBH-positive cells (LC neurons), only one was possibly double stained. Therefore, the vector system we use does not express optogenetic actuators in LC neurons (supplementary Fig. 8). Light did not evoke any consistent effects on neuronal membrane potential or NE release in slices not transduced with astrocyte-targeted AVV or expressing an inert construct (EGFP version CASE12), Supplementary Fig. 9.

Organotypic-cultured brain slices

Experiments were performed in accordance with the UK Animals (Scientific Procedures) Act, 1986 and were approved by the local ethics committees.

Brainstem slice cultures were prepared as described previously61. In brief, Wistar rat pups of either sex (p7–9) were terminally anaesthetized with halothane and immediately decapitated. The brainstem was then removed and bathed in ice-cold dissection medium. Slices were cut at the level of the LC (200 μm thick) in cold (4 °C) sterile dissection solution using a vibrating microtome 7,000 (Campden Instruments, UK). Slices were then plated onto organotypic culture inserts (Millicell-CM, illipore) and supplied with 1 ml per /well plating medium containing the appropriate AVV, diluted to a titre of 108 TU ml–1. Slices were kept at the interface between the feeding medium (pH 7.4) and humidified atmosphere (5% CO2, 37 °C). After 3 days, the plating medium was exchanged for supplemented Neurobasal medium (Gibco, UK), which was subsequently changed twice a week. Slice cultures were allowed to settle for 7–10 days before being used for experimentation to allow AVV-mediated expression to be fully established.

Imaging of astrocytes

Primary cultures of Wistar rat pups (p2) cerebral astrocytes (adapted from62) transduced with AVV.sGFAP.optoβ 2 AR, seeded onto coverslips, were loaded with either Rhod-2AM (30 μM; Invitrogen, UK) or SNARF-5AM (30 μM; Invitrogen, UK) 1 h before visualization. Rhod-2AM was used as a Ca2+ indicator to avoid spectral interference with EGFP. The pH indicator SNARF-5 responds to acidification with a shift in its emission towards shorter wavelengths. Light was collected from two channels set to 550–580 nm (channel 1) and 620–700 nm (channel 2). Ratio of channel 1: channel 2 is indicative of a pH change.

Imaging experiments were carried out at 34 °C under continuous superfusion (150 ml h–1) with pH 7.4 HBS buffer in a tissue chamber mounted on a Leica-SP confocal microscope. Astrocytes were imaged in time-lapse mode (0.5 Hz) using the 543 nm laser line to excite Rhod-2 and SNARF-5. To activate optoβ 2 AR- or ChR2-transduced astrocytes, a 470 nm light-emitting diode-illumination system (Rapp Optoelectronic) coupled to the microscope via the epifluorescence port was employed. Changes of [Ca2+] i in individual cells were assessed by changes in relative fluorescence intensity (F/F 0 ).

Ca2+ imaging and patch clamp of LC neurons

A cultured slice containing LC was transferred to a recording chamber mounted on an upright SP-2 confocal microscope (Leica, Germany) and continuously superfused with HEPES-buffered solution (HBS; in mM: NaCl 137, KCl 5.4, Na 2 HPO 4 0.25, KH 2 PO 4 0.44, CaCl 2 1.3, MgSO 4 1.0, NaHCO 3 4.2, HEPES 10, Glucose 5.5; pH 7.4; 34±1 °C). Current-clamp whole-cell recordings were performed at a 10 kHz sampling rate using an AxoClamp 2B amplifier (Molecular Devices), a 1401 interface and Spike 2 software (both from CED, Cambridge, UK). Recording pipettes were pulled with a vertical puller (Narishige PC-10) to 3–5 mΩ resistance. The pipette solution consisted of (in mM): potassium gluconate 130, HEPES 10, EGTA 11, NaCl 4, MgCl 2 2, CaCl 2 1, ATP 2, GTP 1 and glucose 5. For intracellular L-lactate application, 2 mM L-lactate (pH 7.4) was introduced to the cell by a recording pipette with slightly higher impedance (8–9 mΩ) to slow down L-lactate diffusion and aid visualization of possible time dependent effects of intracellular L-lactate. In these experiments a red-coloured dye was added to the pipette (usually SNARF-5) to confirm successful dialysis of the patched neuron. For Ca2+ imaging, cells were loaded with the Ca2+ indicator Rhod-2 by recording pipette. The intracellular solution used in these experiments contained the following (in mM): potassium gluconate 130, HEPES 10, EGTA 5.5, NaCl 4, MgCl 2 2, CaCl 2 1, ATP 2, GTP 0.5, glucose 5 and Rhod-2 0.5. Changes in [Ca2+] i were assessed by relative fluorescence intensity (F/F 0 ). Rhod-2 was excited using 568 nm laser line, and Rhod-2-emitted fluorescence was sampled at 580–640 nm. For further details see ref. 26. [Ca2+]i responses in LC astrocytes were visualized using a genetically encoded Ca2+ indicator Case12 expressed via an AVV with enhanced GFAP promoter. To boost the expression of the transgenes, the construct employs coordinated co-expression of a chimeric transcriptional activator NFκB-GAL4 (ref. 4).

FCV of extracellular NE

Changes in NE tissue concentrations were measured by FCV using a Millar voltammeter (PD Systems International, UK) and carbon fiber electrodes (CFE; CF10-100 obtained from World Precision Instruments, USA, or produced in house with comparable sensitivity). A symmetric, triangular wave with a range of −0.7 V to 1.3 V (5 ms; 8 Hz) was applied through the CFE to oxidize (at around +0.65 V to +1.0V) and reduce (−0.4 V to −0.6 V) NE (Fig. 5a). Slice cultures containing the LC and transduced with selected AVVs (AVV.sGFAP.optoβ 2 AR, AVV.PRSx8.RFP, AVV.sPRSx8.ChIEF-tdTomato) were immobilized in the recording chamber by a mesh and continuously superfused with HBS (pH 7.4, 34 °C).

The CFE was initially placed in a non-transduced area of the slice and allowed to cycle for 1 h to stabilize the baseline before recording, after which it was transferred to the LC region. Optogenetic stimulation was applied to the LC through an optical fiber (0.7 mm Ø) positioned 10–12 mm above the slice and connected to a PhoxX 445 nm laser (Omicron, Germany).

Before recording each epoch, the background electrochemical signal was subtracted using the ‘refresh’ function of the voltammeter. The resulting Faradaic signal data were acquired and analysed through a Micro1401II interface using Spike2 software (both from CED, Cambridge, UK) with scripts written in house. The NE oxidation peak (between +0.65 V and +1.0 V on the ascending scan) was plotted against time and the integrated NE oxidation in response to L-lactate or light stimulation was determined as V × s. CFEs were retracted from the tissue at the end of experiments and calibrated against NE (0.5 μM) to allow calculation of total release as μM × s. Independently of the electrode sensitivity, we can expect variability in the number of local release sites around any recording position, thus resulting in variation of measured NE volume transmission between recording sites or slices. Therefore data sets including the relevant and comparable control responses were pooled for evaluation of effects.

In vivo experiments

Eleven male Sprague–Dawley rats (300–340 g, 2.5–3 months of age) were anesthetized with urethane (1.6 g kg−1, I.P.). Adequate anaesthesia was ensured by maintaining stable levels of arterial blood pressure and heart rate. The femoral artery and vein were cannulated for measurement of arterial blood pressure and administration of anesthetic, respectively. The trachea was cannulated, and the animal was ventilated with a mixture of 50% oxygen and 50% nitrogen using a positive pressure ventilator (Harvard rodent ventilator, model 683) with a tidal volume of ~2 ml and a ventilator frequency similar to spontaneous frequency (~60 strokes min−1). The body temperature was maintained with a servo-controlled heating pad at 37.0±0.2 °C.

The animal was placed in a stereotaxic frame, and a small hole was drilled in the occipital bone overlying the left LC (AP −9.8 mm; ML −1.2 mm from bregma). For EEG recordings, two small holes were drilled on the left side of the skull above the somatosensory cortex. Stainless steel electrodes were placed in the contact with the cortical surface and secured in place with dental cement.

A three-barrelled glass micropipette (tip size 20–25 μm) was placed in the LC (9.7 mm caudal, 1.15 mm lateral from bregma and 8–8.5 mm ventral from the surface of the skull). The barrels of the micropipette contained L-glutamate (500 mM, pH 7.4), L-lactate (500 mM, pH 7.4) and saline containing 5% of fluorescent beads (Invitrogen). Drugs microinjected from pipettes in small volumes are subject to very rapid dilution and therefore activate a much larger area than the actual volume of injection. In addition, metabolites such as glutamate of L-lactate are taken up by the cells and also eliminated from the tissue into the bloodstream. On the basis of the effective concentrations of L-lactate in vitro established in this work, L-lactate could be applied at concentrations much higher than those of L-glutamate because L-glutamate receptors have high affinity, while the putative L-lactate receptor must have low affinity. Nevertheless we decided not to increase it further to avoid possible complications due to an osmotic effect. Microinjections were made using pressure over a period of 5–10 s and were monitored by observing the fluid level using a dissecting microscope with a calibrated micrometer disk. First, a unilateral microinjection of L-glutamate (40 nl) was made to document the effect of LC activation on cardiovascular variables and EEG. After a recovery period of 30–40 min, L-lactate was microinjected (40 nl) into the same LC site. The sites of microinjections were marked by injection of fluorescent beads, identified Post hoc histologically and mapped using a stereotaxic atlas63. The EEG signal was amplified via a differential amplifier (Digitimer, USA) without additional filtering.

Recordings were processed using a Power1401 interface and analyzed with Spike2 software (CED). Measurements of mean arterial blood pressure and heart rate were taken before and at the peak of the evoked responses and were compared using Student’s paired two-tailed t-test.

The power spectrum of the EEG signal was analyzed using Spike2 software using a custom-written script. 50 (±2) Hz band was excluded to avoid skewing by potential AC interference. L-lactate- and L-glutamate-induced changes in <4 Hz (delta), 4–8 Hz (theta), 8–13 Hz (alpha), 8–30 Hz (beta) and >30 (gamma) power bands were determined and compared within individual experiments.

Post hoc histology

Fixed brains were sectioned and immunostained for dopamine-β-hydroxylase, an established marker for NE neurons. Monoclonal primary antibody was used (MAB308, Millipore, 1:1,000) followed by a goat anti mouse-Alexa 488 (Invitrogen, 1:500).

Drugs

The following drugs were obtained from Tocris: MRS 2179, CNQX, APV, indomethacin, tetrodotoxin, SQ22536, U73122. All other chemicals were from Sigma.

Statistics

Pooled data were expressed as average ±s.e.m. Groups were compared using Student’s t-test, paired or unpaired as indicated. Microsoft Excel was used for data processing. GraphPad Prizm was used to calculate EC50 values for concentration-response curves.