A Method for Activation of Endogenous Acid-sensing Ion Channel 1a (ASIC1a) in the Nervous System with High Spatial and Temporal Precision

Background: Current methods for changing interstitial pH are inadequate to activate ASIC1a in the CNS.

Results: Protons extruded by ArchT elicit ASIC1a currents expressed in same and adjacent cells.

Conclusion: Photostimulation of ArchT in astrocytes activates ASIC1a in nearby neurons leading to train of action potentials.

Significance: This optogenetic-based method applied to awake animals enables examining ASIC1a role in modulating CNS physiology and behavior.

Abstract

Protons activate acid-sensing ion channel 1a (ASIC1a) in the central nervous system (CNS) although the impact of such activation on brain outputs remains elusive. Progress elucidating the functional roles of ASIC1a in the CNS has been hindered by technical difficulties of achieving acidification with spatial and temporal precision. We have implemented a method to control optically the opening of ASIC1a in brain slices and also in awake animals. The light-driven H⁺ pump ArchT was expressed in astrocytes of mouse cortex by injection of adenoviral vectors containing a strong and astrocyte-specific promoter. Illumination with amber light acidified the surrounding interstitium and led to activation of endogenous ASIC1a channels and firing of action potentials in neurons localized in close proximity to ArchT-expressing astrocytes. We conclude that this optogenetic method offers a minimally invasive approach that enables examining the biological consequences of ASIC1a currents in any structure of the CNS and in the modulation of animal behaviors.

Introduction

ASIC1a² is an ion channel activated by extracellular protons in the nervous system. ASIC1a belongs to the large family of sodium channels known as DEG/ENaC (1) whose members share a common structure of three identical or homologous subunits with a large extracellular domain and only two transmembrane segments (2). Because of prominent sodium permeability, opening of ASIC1a depolarizes the plasma membrane contributing to neuronal excitability and Ca²⁺ influx. In the mammalian brain ASIC1a has been implicated in the modulation of synaptic transmission (3), memory, and fear induction (4, 5) and in peripheral nervous system in nociception (6). ASIC1a also contributes to neuronal damage associated with pathological conditions such as ischemia (7) and inflammation (8) of the CNS. However, a number of questions are still under scrutiny about the physiological roles of ASIC1a, its modes of regulation, and implications in pathologies. Most of what is known about its function has been derived from analysis of knock-out mice. The latter is a sound approach, but it is limited by being indirect, subject to compensations by other genes, and the fact that conclusions are drawn from the absence of effects still poorly defined. One of the main hurdles for the advancement of this field has been the lack of means to directly activate ASICs in the brain because of the difficulty in manipulating the external concentration of H⁺ in the brain in a controlled manner. To date, methods used to lower pH, acid perfusion and induction of ischemia, are extreme, irreversible, and unspecific; these maneuvers do not consistently open ASIC1a but rather promote silencing of these channels by inducing steady-state desensitization (9). Hence, there is need for a method to mimic physiological changes in external pH in defined structures of the brain. To meet that challenge we have developed a technique to activate ASIC1a with spatial and temporal precision in the mammalian brain, with minimal invasion of structures and compatible with experiments ex vivo (brain slices) and in conscious animals. To that end, we genetically expressed a light-driven microbial H⁺ pump, ArchT, from the archea Halorubrum sp. TP009 (10, 11), in astrocytes that upon photostimulation extrudes protons acidifying the surrounding external space. We show that such acidification is able to activate endogenous ASIC channels in adjacent neurons; furthermore, the depolarization induced by opening of ASIC1a elicits firing of action potentials.

EXPERIMENTAL PROCEDURES

Cloning of ArchT in pcDNA3.1 and Transfection into CHO Cells

ArchT-GFP was removed from pAAV-CAG-ArchT-GFP obtained from Addgene (plasmid 29777 deposited by E. Boyden) and transferred to the vector pcDNA3.1 to drive transcription of ArchT-GFP by the CMV promoter. This vector was transfected into CHO cells using Lipofectamine 2000 (Invitrogen). Cells were seeded on poly-l-lysine-treated coverslips and examined for fluorescence expression 24 h later.

Construction and High Titer Production of ArchT-GFP and GFP Adenoviral Vectors

The short version of the glial fibrillary acidic protein (GFAP) promoter with the upstream transcriptional amplification modification, p65Gal4BD-mCMV-5×Gal4BS-GfaABC1D, was amplified by PCR from the plasmid pTYF (Addgene plasmid 19976 submitted by S. Kasparov). The PCR product containing the promoter from the pTYF plasmid and the coding region of ArchT-GFP were inserted in pShuttle (Addgene plasmid 16402 submitted by B. Vogelstein). The final construct was sequenced and transiently transfected into primary astrocytes to verify selective expression of the ArchT-GFP protein. The AdEasy system was used to generate adenoviruses as described in detail by Luo et al. (12). Briefly, pShuttle:mCMV/GfaABC1D-ArchT-EGFP was linearized with the restriction enzyme PmeI and electroporated into BJ5183-AD-1-competent Escherichia coli (Agilent Technologies). Cells were seeded on LB/kanamycin plates. 20 small colonies were picked for analysis by restriction endonuclease digestions to confirm correct structure of the insert after recombination between pAdEasy-1 (plasmid carried by BJ5183-AD-1 cells) and the insert of the transfected pShuttle vector. DH10B cells were transformed with the correct recombinant plasmid. The recombinant plasmid was isolated from DH10B cells and digested with restriction enzyme PacI that cuts in a single site. AD-293 cells (Agilent Technologies) were transfected with PacI-digested recombinant adenoviral plasmid using Lipofectamine. After 7–9 days cells were harvested, and viruses were released by several cycles of freezing-thawing-vortexing. Three rounds of amplification generated a large scale preparation of high titer viruses. Purification of viruses was conducted by CsCl gradient centrifugation. Infectious titer was determined by dilution assay immunohistochemical staining using antibodies that detect GFP (13). We obtained preparations with titer ∼10¹⁰ infectious particles/ml. Biohazard level 2 guidelines were applied through out the protocol of viral preparation.

Isolation and Culture of Mouse Cortical Astrocytes

Brains from four P2 mouse pups were isolated; the meninges were removed from cortex hemispheres with fine forceps, and each hemisphere was cut into small pieces followed by digestion in 0.25% trypsin at 37 °C for 30 min in a water-shaker incubator. Tissue pieces were recovered by centrifugation for 5 min at 300 × g. The pellet was mechanically dissociated by vigorous pipetting using a 10-ml plastic pipette and adding 10 ml of DMEM, high glucose + 10% fetal bovine serum + 1% penicillin/streptomycin. The volume was adjusted to 20 ml with medium before plating in two T75 culture flasks. Flasks were incubated at 37 °C in a 5% CO₂ incubator. Medium was replaced every 2–3 days. After 10 days, when cells were confluent, the flasks were shaken at 180 rpm for 30 min on an orbital shaker to remove microglia. Supernatant was discarded, and flasks were shaken at 240 rpm for 6 h to remove oligodendrocytes. Remaining cells, enriched in astrocytes, were removed with 0.25% trypsin-EDTA and transferred to coverslips. Four days later, astrocytes were transduced with adenovirus GFAP promoter-ArchT-GFP or GFAP promoter-GFP.

Stereotaxic Injection of Virus in Mouse Cortex

Four- to 6-week-old mice were anesthetized with isofluorane in O₂ (at ∼0.25–0.5 liter min⁻¹) and transferred to the stereotaxic frame for drilling a <100-μm diameter hole in the skull over the primary visual cortex using stereotaxic coordinates 2.7 mm posterior to Bregma and 2.5 mm lateral to the midline. We injected ∼1.5 μl of virus in cortical layers 1 and 2. After the surgical procedure was completed, analgesia (buprenorphine) was administered for 2 consecutive days according to the Postoperative-Analgesia-Rodents guidelines recommended by the Institutional Animal Care and Use Committee from Yale University. Mice were housed in a biohazard level 2 animal facility for 2 weeks prior to experiments.

Immunohistochemistry

Two weeks after stereotaxic injection of adenoviral vectors in the brain, mice were first anesthetized and intracardially perfused with 0.9% saline and 4% formaldehyde in 0.1 m phosphate buffer, pH 7.4. The whole brain was removed, and serial sections were cut at 15-μm intervals through the thickness of cortex exhibiting GFP in the site of viral expression. Astrocytes were identified by staining with monoclonal antibody anti-GFAP labeled with Alexa Fluor 594, 1:500 dilution in phosphate saline (Chemicon International). Images were captured using an Inverted Zeiss Confocal Microscope (LSM 780).

Fabrication of H⁺-selective Microelectrodes and Measurements of Extracellular pH

We made single barrel H⁺-sensitive surface microelectrodes for measuring changes of H⁺ activity on the surface of cells (14). A thin walled borosilicate glass capillary (GC200TF-4; Warner Instruments LLC) was pulled and fire-polished to a tip of 10–15 μm. Capillaries were baked overnight at 200 °C followed by silanization (bis(dimethylamino)dimethylsilane) (14755; Fluka). The electrode was filled with ∼4 mm of H⁺-ionophore 1 mixture A (95293; Sigma) layered with 40 mm KH₂PO₄, 23 mm NaOH, and 15 mm NaCl, pH 7.0. The H⁺-selective and KCl-filled reference microelectrodes were housed in microelectrode holders both mounted in a dual electrode micromanipulator such that the tips of the two microelectrodes were positioned in close proximity. They were connected, respectively, to channels A and B of a dual channel electrometer. Data were acquired with a digitizer (Iworx 404) connected to a computer.

Patch Clamp Recordings of ArchT and ASIC1a Currents from Cells in Culture

Patch clamp recordings from transfected CHO cells were conducted in the whole cell configuration as described previously (15). Patch pipettes were pulled from PG150T glass (Warner Instruments) to tip diameter of 2–4 μm after heat polishing. Bath solution was 140 mm NaCl, 2 mm KCl, 1.5 mm CaCl₂, 5 mm HEPES, pH 7.3. Pipette solution was 120 mm KCl, 5 mm EDTA, 2 mm ATP, 20 mm HEPES titrated with KOH to pH 7.3. Stimuli of low pH were applied using a Perfusion Fast Step device SF-77B (Warner Instruments) controlled by the data acquisition program Pulse v8.78 according to a protocol of 5-s low pH and recovery for 10 s at pH 7.4. Recordings were made using an EPC-9 amplifier and the Pulse acquisition program v8.78 (HEKA Electronic). Experiments were conducted at room temperature. Light source was a mercury 100-W lamp installed in an inverted Nikon Eclipse TE200 microscope. For visualization of GFP, light was passed through a 430/24-nm excitation filter, and illumination or ArchT was through a 580/30 excitation filter.

Light intensity at 580 nm, corresponding to ArchT absorption maximum, was measured at the object plane using an optical power meter (PM160; Thorlabs). The intensity of illumination was calculated with the ×40 magnification objective lens of the microscope by measuring the radius of the illuminated area; we then divided the total light intensity by the area.

Preparation of Brain Slices, Photostimulation, and Electrophysiological Recordings

Two to 3 weeks after viral injection, mice were anesthetized by injection of 60 mg/kg ketamine and 2 mg/kg xylazine followed by immediate decapitation. Brains were quickly removed and transferred to oxygenated (95% O₂/5% CO₂) ice-cold Ca²⁺-free artificial cerebrospinal fluid (124 mm NaCl, 3 mm KCl, 1.3 mm MgSO₄, 26 mm NaHCO₃, 1.25 mm NaHPO₄, 20 mm glucose, 2 mm CaCl₂; ∼310 milliosmoles, pH 7.4, when bubbled with 5% CO₂. 300-μm-thick coronal slices were cut with a vibrotome (VT1000 S; Leica Microsystems) and allowed to recover for 30–45 min at room temperature. Slices were transferred to the recording chamber of the electrophysiology rig and superfused (1–2 ml/min) with oxygenated artificial cerebrospinal fluid supplemented with 50 μm [d,l]-2-amino-5-phosphonovalerate and 10 μm 6,7-dinitroquinoxaline-2,3-dione from Tocris (Ellisville, MO. Patch pipettes (4–8 megohms) pulled from borosilicate glass were filled with internal solution containing 144 mm potassium gluconate, 3 mm MgCl₂, 0.2 mm EGTA, 10 mm HEPES, pH 7.3 (290 milliosmoles). Electrophysiological recordings were performed in current clamp mode at room temperature using a patch clamp amplifier (Axopatch 200B; Axon). Data were filtered at 5 kHz and digitized at 50 kHz using an acquisition board (Digidata 1440A/pCLAMP10; Molecular Devices).

Optical stimulation was performed using the OptoLED system (Cairn Research, Faversham, UK), consisting of a 590-nm, 3.5-W LED mounted on a BX51WI microscope (Olympus) equipped with infrared differential interference contrast optics (900 nm) and epifluorescence. Optogenetic stimulation was applied for 3 s as pulses of 12.5-, 25-, or 50-ms width at 20 Hz. The illuminated area was approximately 2 mm, which corresponds to the area of the slice visualized using a ×40/0.8 numerical aperture water immersion objective.

Whole cell recordings of ASIC currents of neurons from slice preparations were conducted as described previously (16).

Chemicals were from Sigma unless indicated otherwise. Psalmotoxin-1 (PcTx1) was purchased from Abcam.

RESULTS

Acidification of the External Medium by Illumination of ArchT-expressing Cells

Central to the idea of using light to activate native ASIC1a channels in the nervous system is to demonstrate that ArchT is able to acidify the extracellular compartment. We therefore measured changes of cell surface pH with H⁺-selective microelectrodes constructed as indicated under “Experimental Procedures.” Calibration of the pH microelectrode was conducted by replacing the bath with solutions of known pH and measuring the voltage changes with a high impedance dual channel differential electrometer constructed in house. One channel from the electrometer acquired data from a headstage connected to the H⁺-selective microelectrode whereas the other channel acquired data from the KCl-filled reference electrode. The difference between the outputs from the two channels is proportional to the H⁺ activity of the solution as plotted in Fig. 1A.

FIGURE 1.

Activation of ArchT in transfected cells acidifies the external pH. A, calibration of an external pH-selective microelectrode filled with an H⁺-ionophore 1 mixture. A reference electrode was placed close to the pH microelectrode. Voltages were acquired with a high impedance dual channel differential electrometer. The slope of the curve corresponding to the Nernst potential for H⁺ is shown: −58 mV/pH unit. B, examples of cell surface pH recorded from CHO cells transfected with pcDNA3.1-ArchT-GFP. Traces are voltage changes recorded with the external pH microelectrode positioned close to the cell surface of a cell illuminated with light 580 ± 30 nm, power 3 mW/mm². Yellow shade represents the illumination time. Left of the traces is a pH scale constructed from the calibration shown in A. Illumination induces an external pH change of ∼0.2 pH unit. Data were acquired via a digitizer (iWorx/214) connected to a PC computer, visualized, and recorded using LabScribe software.

To measure pH changes induced by photoactivation of ArchT, CHO cells seeded on coverslips were transfected with ArchT-GFP. Cells expressing high intensity fluorescence at the plasma membrane were selected for experiments. The tip of the pH microelectrode was placed in close proximity to the cell surface of bright fluorescent cells guided by a micromanipulator. The pH of the chamber solution was 7.3 buffered with 5 mm HEPES. Perfusion of the chamber was stopped during measurements of external pH to slow diffusion of protons. Cells were illuminated with 530-nm light for 10 s (yellow shade in Fig. 1B). Traces represent voltage changes transformed to pH units according to the calibration of the H⁺-selective microelectrode. There was a slight variability in the response which we attributed to small changes in the position of the electrode tip to the cell surface as this measurement is highly sensitive to distance from the electrode tip to the cell membrane. The pH scale on the left of the traces reads 0.2 pH unit of acidification. However, these measurements underestimate the actual maximal pH change due to rapid diffusion of H⁺ to the bath solution and to the slow time response of the microelectrode. Together, the experiments indicate that light activation of ArchT leads to significant acidification of the extracellular solution and support the idea that ArchT could be used as a rapid switch to reducing external pH in selected structures of the nervous system.

Activation of ASIC1a in Cultured Cells by Light-driven Secretion of Protons

In the following experiments we tested whether protons extruded by ArchT activate ASIC1a channels when both proteins are expressed in cultured cells. CHO cells were seeded on coverslips and transfected with ArchT-GFP alone or co-transfected together with ASIC1a fused with mCherry at the C terminus. 24–32 h after transfection most cells exhibited robust fluorescence localized primarily at the plasma membrane, although variable degrees of intracellular fluorescence were also detected in a small fraction of cells (Fig. 2A). Coverslips were transferred to the microscope chamber of a patch clamp setup, and cells expressing high intensity of GFP in the plasma membrane were selected for measurements of ArchT activity. The latter was determined as the current evoked by illuminating cells with pulses of 530-nm light at an intensity of ∼5 mW/mm² for 0.4 s provided by a mercury lamp while the membrane potential was held at −40 mV. Fig. 2B shows that light pulses (orange bars over current traces) consistently induced outward currents, average magnitude 50 ± 10 pA/cell, as expected for extrusion of H⁺ by ArchT and with little or no decay. Current traces also show that as the extracellular pH was decreased, from 7.4 to 4.0 and 2.5, the magnitude of the outward current decreased, again consistent with protons being pumped out by ArchT.

FIGURE 2.

Activation of ASIC1 by illumination of ArchT in cultured cells. A, fluorescent image of cells transfected with ArchT-GFP. B, whole cell currents of a representative ArchT-expressing cell evoked by illumination with 580 ± 30 nm light (3 mW/mm²) for 0.4-s pulses (orange bars). The magnitude of the outward currents decreased as the pH of the bath solution was decreased from 7.4 to 6.5, 4.0, and 2.5. Membrane voltage was held at −40 mV. C, illumination of a cell co-expressing ArchT and human ASIC1a evoking a biphasic current. Immediately after photoactivation of the outward current there is a transient component of inward current that decays in subsequent light pulses (second and third traces shown in gray). D, protocol identical to that shown in B but with 100 μm amiloride in the bath eliminating the inward component of the currents. E, after washout of amiloride, rapid application of three consecutive pH 6.5 stimuli evoking only inward currents with properties distinctive of hASIC1a (first trace in red with second and third shown in gray).

We next examined cells co-transfected with ArchT and human ASIC1a. Fig. 2C shows whole cell currents of a patched cell expressing both proteins and illuminated by three consecutive light pulses. Photostimulation induced rapid outward current as in the previous experiment, but rather than being sustained, the current transiently decreased. The transient decrease in current consists of simultaneous activation of ASIC1a expressed in the same cell. That the inward component of the currents is indeed mediated by ASIC1a was confirmed by application of 100 μm amiloride to the bath solution, which suppressed the inward currents without affecting the outward component (Fig. 2D). Furthermore, attenuation of the inward currents upon consecutive light pulses administered at a frequency of 5 Hz, shown in gray in Fig. 2C, is consistent with the slow recovery from desensitization characteristic of hASIC1a (16). Fig. 2E illustrates this phenomenon where a cell expressing hASIC1a was exposed to three consecutive pH 6.5 stimuli of 0.4-s duration at intervals of 0.2 s. The inward currents shown in Fig. 2E have more rapid kinetics than those shown in Fig. 2C because the rise in the concentration of protons is much faster when cells are stimulated by rapidly changing the external solution from pH 7.4 to 6.5 compared with the rise of H⁺ concentration generated by ArchT activity. Together these experiments provide experimental evidence that ASIC1a is activated by illumination of ArchT pumps expressed in proximity to the channels.

Expression of ArchT in Cultured Primary Astrocytes and in Mouse Brain

To activate endogenous ASIC1a it is essential to express high levels of ArchT in cells of the nervous system. We chose to express ArchT in astrocytes rather than in neurons to avoid the hyperpolarization of neurons that arises from H⁺ extrusion. Such hyperpolarization would interfere with the cellular responses induced by opening of ASIC1a, namely a depolarization of the membrane potential because of Na⁺ ions influx through ASIC1a channels.

To target astrocytes selectively we constructed an adenoviral vector that expresses ArchT-GFP driven by the astrocyte-specific promoter for GFAP. We used a short version of the GFAP promoter that is 690 bp in length (GfaABC₁D) (17) linked to a two-step transcriptional amplification strategy (TSTAS) (18) that increases expression level of the transgene up to 5-fold. TSTAS relies on a positive feedback loop that operates by co-expression of a chimeric transcriptional enhancer Gal4-NFκB (19). The latter is a fusion protein consisting of the transcriptional activation domain of the NFκB p65 protein fused to the DNA-binding domain of Gal4 protein from yeast. The promoter GfaABC₁D operates bidirectionally driving expression of both ArchT-EGFP and the enhancer. The mini-CMV mammalian promoter alone, mCMV, drives weak transcription but when located upstream and in opposite orientation to an efficient promoter drives coordinate transcriptional activity in both directions (20). Transcription of the enhancer then boosts level of expression of ArchT-GFP, which is necessary to achieve significant extracellular acidification. Fig. 3A illustrates a schematic of the inserts of two pAdEasy shuttle vectors: GFAP/ArchT-GFP and the control vector identical to the previous one but encoding only GFP. Primary astrocytes were isolated from neonate mouse brain cortex and infected with GFAP promoter-ArchT-GFP adenovirus. Three days later cells were fixed and stained with Alexa Fluor 594-conjugated GFAP monoclonal antibody. Fig. 3B shows astrocytes as thin flat cells spreading over the coverslip. The image of the whole thickness of the cells shows ArchT-GFP expressed mostly at the plasma membrane (there are a few intracellular aggregates in astrocytes) and GFAP as cytosolic bundles running parallel to the plasma membrane.

FIGURE 3.

Expression of ArchT-GFP in mouse astrocytes. A, diagram of the expression cassette. The compact glial fibrillary acidic protein promoter GfaABC1D (mGFAP) and the minimal core promoter derived from the human cytomegalovirus (mCMV), both shown in red, are in opposite orientations and are followed by the coding regions of the two proteins to be expressed: the enhancer chimeric protein GAL4BD-NFκBp65 that binds to five repeats of the GAL4 binding sequence (5×GAL4 BS) and ArchT-EGFP. WPRE, woodchuck hepatitis post-transcriptional regulatory element for enhancement of gene expression. LITR and RITR, left and right inverted terminal repeats, respectively. Shown below are the same adenoviral construct but expressing only GFP. B, primary mouse astrocytes grown in culture transfected with adenovirus expressing ArchT-GFP driven by GFAP promoter. Endogenous GFAP was stained with a monoclonal antibody conjugated with Alexa Fluor 594. C, confocal images of a 20-μm-thick brain slice of brain cortex fixed with 4% paraformaldehyde prepared 2 weeks after stereotaxic injection of ArchT-GFP adenoviral vector. Slices were stained with monoclonal anti-GFAP conjugated with Alexa Fluor 594. Images were obtained in an inverted Zeiss Confocal Microscope (LSM 780). The three channels (DAPI, GFP, and Alexa Fluor 594) were scanned separately and merged using Zen software.

Adenoviruses were injected into the superficial layers of the primary visual cortex of mouse brain by stereotaxic surgery. Approximately 1.5 μl of viral solution, titer 10¹⁰ transforming units/ml, was injected in each site. Two weeks after injection, brains were harvested and examined for expression of ArchT using immunochemical staining. A monoclonal antibody against GFAP was used to identify astrocytes. Fig. 3C shows representative images of slices transduced by the adenoviral vector. Only cells positive for GFAP expressed GFP, indicating that the vector induces expression of ArchT exclusively in astrocytes. The image also shows that ArchT-GFP localizes primarily to the plasma membrane whereas GFAP is in the cytosol of astrocytes.

Photostimulation of ArchT Activates ASIC1a in Brain Slices

Brain slices from mice injected with ArchT-GFP adenoviruses in brain cortex were used for testing activation of endogenous ASIC1a. The area of viral expression was identified by fluorescence emitted by GFP upon light excitation passed trough a 430/24-nm filter. Excitation time was kept short to minimize activation of ArchT. The soma of pyramidal neurons surrounded by fluorescent astrocytes were patched in the whole cell configuration and examined under current clamp. Current injection was adjusted to attain a membrane potential of −60 mV. We then determined the degree of illumination of ArchT (590-nm light supplied by a 3.5-W LED) required to eliciting action potentials by increasing the duration of the light pulses. Upon illumination, membrane depolarization was observed with a 230 ± 100-ms delay and persisted for 180 ± 60 ms after the stimulus was terminated. The variability in the initiation and termination of depolarization was most likely related to the position of the patched neuron with respect to ArchT-expressing astrocytes, because distance is expected to decrease and delay the peak change of extracellular pH. Fig. 4A shows the response of a typical neuron to increasing illumination intensity. Illumination time of 3 s with 12.5-ms pulses did not reach the threshold of activation, −43 ± 5 mV, in this cell. Increasing the duration of light pulses to 25 and 50 ms (2.43-mW/mm²) evoked trains of action potentials in 33 and 68% of examined cells (n = 34). Similar experiments conducted in the presence of the spider toxin PcTx1, which is a specific inhibitor of ASIC1a, or in slices previously injected with adenoviral vector expressing only GFP (n = 12), elicited neither depolarization nor spikes upon illumination (Fig. 3B). Neurons in slices injected with GFP adenovirus responded to a puff of external solution, pH 5.0, by inducing transient inward currents, and PcTx1 inhibited those currents (Fig. 4C). Therefore, the results are consistent with ArchT-mediated acidification of the interstitium surrounding astrocytes with subsequent activation of ASIC1a in neurons. These events in turn lead to induction of action potentials. Although neurons in the CNS express more than one type of ASIC channel, the most likely subunit underlying the observed depolarization is ASIC1a. This assertion is based on the facts that ASIC1a is the most abundant isoform expressed in central neurons, it has the highest sensitivity to H⁺ (pH_50A 6.8), and is selectively inhibited by PcTx1 (21).

FIGURE 4.

Photoactivation of endogenous ASIC1a in brain slices. A, representative responses of pyramidal neurons from mouse visual cortex show expression of ArchT in astrocytes. Optical stimulation of astrocytes with amber light supplied by a 590-nm LED (orange trace below voltage recordings) depolarized the membrane potential of neighboring neurons detected by an intracellular electrode. Increasing the light stimulus, i.e. lengthening the pulses from 12 to 50 ms, evoked larger depolarizations and triggered action potentials. The response was completely blocked in the presence of the ASIC1a-specific inhibitor PcTx1. B, a similar experiment conducted in slices previously injected with adenovirus expressing only GFP did not respond to illumination. C, current traces of pyramidal cells from the same brain slice but obtained in the voltage clamp configuration were evoked by rapid changes in the external pH solution from 7.3 to pH 6.9 or pH 5.0. PcTx1 in the bath prevented proton-induced currents. Time between stimuli was 60 s. D, depolarization and train of action potentials are attenuated when the stimulus is applied at short intervals. Full recovery was observed at intervals ≥50 s.

It is widely acknowledged that currents from mammalian ASIC1a decrease following consecutive stimuli because of slow recovery from desensitization after an acid stimulus; this is shown in Fig. 2, C and E. Recovery from desensitization takes place at pH equal or higher than 7.3 and follows two exponentials with mean times τ_Dshort ≤0.5 s and τ_Dlong 229 s (15). We therefore examined the magnitude of a second response to photostimulation applied either 5 or 50 s apart. As shown in Fig. 4B the response to a second stimulus was attenuated when administered after 5 s but recovered almost completely after 50 s. Although measurements of interstitial pH in brain slices were out of the scope of this work, we deduce from the results that the external pH returns to baseline during the interval between stimuli, thereby preventing steady-state desensitization of ASIC1a by prolonged exposure to low pH. Another factor that may contribute to maintaining the level of response is the fact that interstitial acidification induced by ArchT photostimulation is mild, thereby only a small fraction of ASIC1a channels is activated leaving a large pool of resting channels available to the next pH stimulus. Indeed, when we examined H⁺-induced whole cell currents in pyramidal neurons by rapidly changing the perfusion solution from pH 7.3 to 6.9 we observed transient inward currents of average amplitude 150 ± 30 pA whereas a supramaximal stimulus with pH 5.0 applied 50 s after the first one induced currents of 420 ± 59 pA (Fig. 4C). This confirms that ArchT lowers the external pH to ∼6.9 and activates only a small population of ASIC1a that is sufficient to depolarize neurons to the threshold of action potentials.

DISCUSSION

The absence of suitable means to activate ASIC1a specifically and precisely in the brain prompted us to examine the applicability of optogenetic tools for this purpose. We show that the light-driven proton pump ArchT, when stimulated by light of the appropriate wavelength and intensity, activates ASIC1a channels expressed in the same cell membrane (cis configuration) or in adjacent cells (trans configuration). Specifically, we tested whether expression of ArchT in astrocytes activates endogenous ASIC1a in superficial neurons of mouse cortex. We show that the approach works in brain slices and can be extended to conscious animals paving the way to examining the biological functions of ASIC1a in vivo. This approach is thus superior to inducing ischemia or injecting channel openers (6) as these maneuvers are much more difficult to control and are not rapidly reversible.

As all new techniques this one also raises concerns, a few of which we discuss here. One concern relates to specificity, i.e. interstitial H⁺ ions modulate activity of other channels in the CNS in addition to ASIC1a. Although most proteins are affected by pH, only a few use external protons as specific agonists or modulators; examples are K⁺ channels of the two-P-domain family (TASK1 and TASK3) (22) and TRPV1 (23). External protons inhibit TASK channels in the pH range 6.0–8.0; therefore, these channels are likely to close upon the acidification induced by ArchT. Simultaneous closing of TASK channels and opening of ASIC1a in the same neuron would produce a stronger depolarization than if only one type of these channels was expressed, thereby leading to an additive effect. The expression pattern of the TASK channels in the CNS is, however, more restricted than that of ASIC1a. In the mouse, TASK1 is highly abundant in cerebellar granular cells followed by the raphe nuclei, olfactory bulb granule cells, and brain stem trigeminal nuclei (24). Very little is expressed in thalamus and neocortex as shown in our experiments of superficial pyramidal cells of the visual cortex where no depolarization was detected in the presence of ASIC1a inhibitors or when GFP alone was expressed in astrocytes. It is even less likely that ArchT would activate TRPV1 channels in vivo because of their limited expression (caudal hypothalamus and discrete nonpyramidal hippocampal neurons (25)), but most importantly, because the pH for TRPV1 activation is too low (pH <6.0) to be reached by illumination of ArchT.

However, activation of ArchT is expected to transiently hyperpolarize astrocytes; the functional impact of such hyperpolarization is also unknown to date (26, 27). One could posit that it might affect transport of ions and small molecules with consequences for ASIC1a recovery from desensitization as astrocytes are the most likely cell type to reabsorb protons from the extracellular space thereby restore the pH to baseline level. The recovery of the ASIC1a response 50 s after photostimulation makes it unlikely that short illumination produces cell damage or significantly disrupts the astrocyte function under our experimental conditions.

It is also important to underscore that with this experimental method our aim was not to address the question of what is the natural source of protons that activates ASIC1a in the brain under physiological conditions or whether another endogenous substance, yet to be identified, acts as the preferred agonist. We only exploited the fact that by manipulating local proton concentrations, which is the most widely acknowledged way to activate ASIC1a, it is possible to control opening of the channel optically thus enabling to probe with greater detail the role of these channels in modulating neuronal activity. In summary, this novel tool based in optogenetics assures a high degree of control of ASIC1a activity with minimal invasion, making it ideal for behavioral studies of conscious animals and further our current understanding of the physiology of these channels in the mammalian CNS.