Application of an Optogenetic Byway for Perturbing Neuronal Activity Via Glial Photostimulation

Application of an optogenetic byway for perturbing neuronal activity via glial photostimulation

Takuya Sasakia,1, Kaoru Beppua,b,1, Kenji F. Tanakab,c,2, Yugo Fukazawaa,b,d,3, Ryuichi Shigemotoa,b,e, and Ko Matsuia,b,f,4

Divisions of aCerebral Structure and cNeurobiology and Bioinformatics, National Institute for Physiological Sciences, Okazaki 444-8787, Japan; bDepartment of Physiological Sciences, Graduate University for Advanced Studies (Sokendai), Okazaki 444-8787, Japan; and dCore Research for Evolutional Science and Technology, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0075, Japan; eSolution-Oriented Research for Science and Technology and fPrecursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi 333-0012, Japan

Edited by Charles F. Stevens, The Salk Institute for Biological Studies, La Jolla, CA, and approved October 30, 2012 (received for review August 6, 2012)

Dynamic activity of glia has repeatedly been demonstrated, but if up” and interact functionally with PCs (Fig. S1). Release of such activity is independent from neuronal activity, glia would not transmitter from glia (gliotransmitter) has been demonstrated in have any role in the information processing in the brain or in the several brain areas (12–16) and we sought to address whether such generation of animal behavior. Evidence for neurons communicating gliotransmitter could mediate glia-to-neuron communication in with glia is solid, but the signaling pathway leading back from glial- the cerebellum. to-neuronal activity was often difficult to study. Here, we introduced To deal with this question, we need a method to selectively a transgenic mouse line in which channelrhodopsin-2, a light-gated activate the glia. Conventional electrophysiological techniques, cation channel, was expressed in astrocytes. Selective photostimula- such as extracellular electrical stimulation, inevitably stimulate tion of these astrocytes in vivo triggered neuronal activation. Using both neuron and glia. Thus, gliotransmitter release could have slice preparations, we show that glial photostimulation leads to been unintentionally evoked in previous studies but its effect overlooked. Direct intracellular stimulation of glia is also difficult release of glutamate, which was sufficient to activate AMPA re- as the input resistance of these cells is low and the depolarization ceptors on Purkinje cells and to induce long-term depression of fi generated by the somatic electrode will not propagate through the parallel ber-to-Purkinje cell synapses through activation of metab- complex glial structures. We used optogenetics to circumvent this otropic glutamate receptors. In contrast to neuronal synaptic vesicular problem (17). An important advantage of the optogenetic tools, NEUROSCIENCE release, glial activation likely causes preferential activation of extra- such as channelrhodopsin-2 (ChR2), a light-gated nonselective synaptic receptors that appose glial membrane. Finally, we show that cation channel, is its cell type specificity. Although glia-specific neuronal activation by glial stimulation can lead to perturbation of expression of ChR2 has been achieved using viral vectors in pre- cerebellar modulated motor behavior. These findings demonstrate vious studies (12, 18), we took an approach to generate a trans- that glia can modulate the tone of neuronal activity and behavior. genic mouse line in which highly-sensitive mutant [C128S (19)] of This animal model is expected to be a potentially powerful approach ChR2 was selectively expressed in astrocytes including BGs using to study the role of glia in brain function. the tetracycline transactivator (tTA)–tet operator (tetO) system (20). In contrast to viral vectors, the transgenic lines allow stable cerebellum | Bergmann glia | gliotransmitter | plasticity | c-fos and reproducible expression of transgenes in animals. In this study, we demonstrate the presence of glia-to-neuron communi- uch of the brain function is often perceived as being ach- cation in the transgenic animals both in vitro and in vivo. These Mieved solely by neuronal activity in contemporary science. transgenic tools would be indispensable for investigating in- However, the brain is actually occupied mostly by another type of tercellular signaling in the brain. cells, the glia. Their role was classically considered to be re- Results stricted to the maintenance of neuronal survival and functioning, + such as trophic support, uptake of glutamate, and removal of K Optogenetic Stimulation of Bergmann Glial Cells Drives Neuron Activity. from the extracellular space (1), but rapid and dynamic activity To selectively stimulate the glia, we generated two lines of mice of glia correlated with behavior and cognitive functions has re- (20); one in which the expression of tetracycline-controlled tTA is cently been shown (2, 3). However, demonstration of the causal driven by megalencephalic leukoencephalopathy with subcortical link between glial activity and neuronal activity is required to cysts 1 (Mlc1)-promoter, which has been proven to be astrocyte fi accept the possibility that glia can indeed participate in the in- speci c (21), and the other in which tetO is connected to the ex- formation processing in the brain. pression of C128S mutant of ChR2 (19) fused with enhanced yellow fl In the cerebellum, there are direct and rapid mechanisms for uorescent protein (EYFP). By obtaining bigenic [Mlc1-tTA::tetO- neurons to communicate with glia. Stimulation of the parallel ChR2(C128S)-EYFP] mice, strong EYFP expression was found in A fibers (PFs), the axons of granule cell neurons (GCs), triggers BGs in the cerebellum (Fig. 1 ). A point mutation (C128S) of the glutamate release from synaptic sites and additional ectopic sites, ChR2 causes prominent increases in photosensitivity as well as evoking an inward current in the adjacent major cerebellar astrocytes, the Bergmann glial cells (BGs), with components due to both + activation of Ca2 -permeable AMPA receptors (AMPARs) and Author contributions: K.M. designed research; T.S., K.B., and K.M. performed research; electrogenic uptake of glutamate (4–6). Computational modeling K.F.T., Y.F., and R.S. contributed new reagents/analytic tools; T.S., K.B., and K.M. analyzed has suggested that such synaptically induced activity would cause data; and T.S., K.B., and K.M. wrote the paper. strikingly large depolarization (up to 40 mV) of the BG membrane The authors declare no conflict of interest. processes local to the activated sites (7). Furthermore, PF burst This article is a PNAS Direct Submission. 2+ activity elicits transient and local Ca increases in BG processes 1T.S. and K.B. contributed equally to this work. via AMPA and ATP receptor-mediated mechanisms (2, 8–11). 2Present address: Department of Neuropsychiatry, School of Medicine, Keio University, Despite the accumulating evidence of neuron-to-glia communi- Tokyo 160-8582, Japan. cation in the cerebellum, if the communication between these cells 3Present address: Department of Anatomy and Molecular Cell Biology, Nagoya University is a one-way relationship, the glia would only be a “listener” and it Graduate School of Medicine, Nagoya 466-8550, Japan. would not be able to actively participate in a layer of information 4To whom correspondence should be addressed. E-mail: [email protected]. processing. The close anatomical apposition of BGs to neighboring This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Purkinje cells (PCs) suggests the possibility that BGs could “speak 1073/pnas.1213458109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1213458109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 alteration in the photocycle, enabling a step-like control of the of yellow light; however, the remaining current required >30 s to photocurrent by blue and yellow light (19). To validate the selective return to baseline (Fig. 1B and Fig. S3A). We were uncertain expression of ChR2(C128S) in glial cells, whole-cell patch-clamp about the cause of this slowly developing current; therefore, we recordings were performed in acute cerebellar slices in the presence generated parvalbumin (PV)-tTA::tetO-ChR2(C128S)-EYFP of a mixture of pharmacological agents that silence neuronal ac- mice in which EYFP expression was confirmed in PCs (Fig. S3B). tivity (Fig. 1B). Current responses to photostimulation were In this mouse line, delivery of the blue light produced a constant detected only in BGs (n = 24), but not in PCs (n = 10), GCs (n = inward current in PCs, which was rapidly terminated with the n = yellow light pulse. We assumed that the cause of the additional 10), and stellate/basket cells (SC/BCs) ( 11). Furthermore, si- + current in BGs is the high conductance of BGs to K making multaneous in situ hybridization for tTA and immunohistochemis- + them sensitive to changes in extracellular K concentrations try for brain lipid-binding protein (BLBP) confirmed the specificity + (22). As expected, 5 mM Ba2 and 5 mM 4-aminopyridine (4- of transgene expression in BGs (Fig. S2A) with modest expression + in the protoplasmic astrocytes in the GC layer (GL) (Fig. S2B). AP), K channel blockers, inhibited a large fraction of the slow component, whereas the fast component remained intact (Fig. These results demonstrate that, in our transgenic mice, ChR2 ex- C D pression is restricted to glial cells. S3 and ). These results indicate that the fast component is likely attributable to ChR2 opening itself and the slow component In BGs, photostimulation with a blue light produced an in- + wardly developing current that was terminated partially by a flash to extracellular K change. The size of both of these components developed in an age-dependent manner (Fig. S3 E–G). Our aim was to assess whether glia-to-neuron communication can be elicited by photostimulation of glia. To evaluate this in vivo, fi A Mlc1-tTA::tetO-ChR2 an optic ber was placed just on the skull above the cerebellum of EYFP the transgenic mouse (Fig. 1C). While the mouse moved freely in a home cage, a single photostimulation (200-ms blue and 200-ms P ChR2-EYFP yellow with an interval of 500 ms) was applied (Fig. 1D). After killing the mouse, in situ hybridization for c-fos mRNA, an im- DIC mediate-early gene as a marker of neuronal activation, was performed (Fig. 1E). At the region directly beneath the optic fiber, strong c-fos expression was observed in ∼50% of the PCs and SC/ BCs, and a subset of GCs (Fig. S2C). Expression of c-fos was not observed in nonphotostimulated ChR2(+) mice or in photostimulated ChR2(−)mice(Fig. S2 D and E). The microglial cell distribution and morphology appeared normal (Fig. S2F). 50 µm

B Bergmann glia Purkinje cell Granule cell Stellate/Basket cell Identity of the Gliotransmitter and Mechanism of Release. Even Blue though only the glia was photostimulated, in the absence of Yellow pharmacological agents, c-fos was elevated in neurons and a burst of firing activity could be elicited in PCs in slices (Fig. S4A). Al- + 200 pA though elevated K could be one of the mechanisms underlying 5 s neuronal excitation, we examined whether any other substance is C D released following glia photostimulation. The receptors expressed 200 ms on PC membrane were used as biosensors to detect the released Blue substance in acute cerebellar slices. Neuronal transmitter release was inhibited by applying 0.5 μM tetrodotoxin (TTX) and 100 μM 500 ms + + Cd2 , to block action potentials and voltage-gated Ca2 channels, Yellow respectively, and 100 μM pictrotoxin (PIC) was applied to block 200 ms GABAA receptors. Glia photostimulation in slices from Mlc1- tTA::tetO-ChR2(C128S) mice produced an inward current in the E recorded PC (Fig. 2A), suggesting that the receptors on PCs were Optical fiber activated. In control condition, we noticed that glia-photostimulated PC currents declined with time during repetitive photostimulation (Fig. S5C). The direct rundown of ChR2 activation was also observed in PCs from PV-tTA::tetO-ChR2(C128S) mice and in BGs from Mlc1-tTA::tetO-ChR2(C128S) mice (Fig. S5 A B 200 µm and ). These results imply that the rundown of the glia-photostimulated PC currents is, at least in part, due to the rundown of ChR2 activation. The following data were normalized to the amplitudes of currents in PCs recorded in the presence of TTX + and Cd2 at each corresponding period after establishment of the 1 mm whole-cell recordings. 50 µm We next sought to identify the transmitter. Because glia-photostimulated PC currents remained intact in the presence of PIC (Fig. Fig. 1. Glia photostimulation in vivo leads to neuronal activity. (A) Crossing S4B), GABA is not likely to be the major transmitter (23). Appli- two lines of mice [Mlc1-tTA and tetO-ChR2(C128S)-EYFP] yielded mice in which cation of AMPAR antagonist, 10 μM 2,3-dioxo-6-nitro-1,2,3,4- ChR2(C128S)-EYFP was selectively expressed in Mlc1-positive astrocytes in- tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (Fig. S4C) cluding cerebellar BGs. The lower panels show a differential interference con- or 100 μM GYKI53655 (P < 0.001, n = 6; Fig. 2A), entirely abol- trast (DIC) image and a confocal stack image of EYFP in an identical region of μ the parasagittal section of the cerebellum. (B) Typical current traces in response ished the glia-photostimulated PC current, whereas 20 Mcyclo- thiazide (CTZ), an AMPAR desensitization inhibitor, effectively to photostimulation recorded from a BG, PC, GC, and SC/BC (similar results from fi D 24, 10, 10, and 11 cells, respectively). Recordings were performed in the pres- ampli ed the current (Fig. S4 ). If the glutamate concentration + ence of TTX, Cd2 , PIC, and NBQX to silence neuronal activity and extract ChR2 rise is slow, the AMPARs would become desensitized, whereas activity. (C and D) Sequence of light applied in vivo via optical fiber placed on CTZ would be able to cancel this desensitizing effect. The kinetics top of the skull. (E)c-fos mRNA (10 min after the photostimulation) was in- of the slowly developing PC current was mimicked by local pressure creased in PCs (arrowheads), GCs, and SC/BCs (n = 4animals). injection (puff) of exogenous glutamate (Fig. S4E). Addition of

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1213458109 Sasaki et al. Downloaded by guest on September 28, 2021 2+ 1.5 increase, which is detected as currents in the recorded PCs. It is A TTX,Cd ,PIC +GYKI 2+ Blue 1 known that Ca -permeable (GluR2-lacking) AMPARs are not Yellow expressed on PCs but are expressed on BGs and SC/BCs and these 0.5 receptors could be selectively blocked by 1-naphthylacetyl sper- 0 Norm. amp. mine (NASPM). The glia-photostimulated PC current was in- PC 50 pA sensitive to 100 μM NASPM (Fig. S6B); thus, this alternative -10 -5 0 5 10 15 2+ 5 s (min) pathway mediated by Ca -permeable AMPARs is unlikely. Several pathways for glutamate release from glial cells are + 2+ plausible (26): (i) reversal of uptake by Na -dependent glutamate B TTX,Cd ,PIC +TBOA 10 transporters, (ii) functional unpaired connexin hemichannels, (iii) Blue 2+ Yellow volume-sensitive anion channel opening, and (iv)Ca -dependent 5 exocytosis. It has been suggested that the reversal of glutamate 100 pA transporter can be blocked by extracellular TBOA (27); however, PC Norm. amp. 0 5 s TBOA failed to inhibit glia-photostimulated PC current but rather -10 -5 0 5 10 15 B μ (min) enhanced it (Fig. 2 ). In the presence of 100 M carbenoxolone, one of the most widely used gap junction/hemichannel blockers, 2+ 1.5 the current response remained normal (Fig. S7A), ruling out the C TTX,Cd ,PIC +DIDS Blue 1 contribution of hemichannel opening. Blocking swelling activated Yellow anion channels with 100 μM 5-nitro-2-(3-phenylpropyl amino) 0.5 benzoic acid (NPPB) did not alter the glia-photostimulated PC 0 B ’ ’ PC Norm. amp. current (23) (Fig. S7 ). 4,4 -Diisothiocyanostilbene- 2,2 -disul- 50 pA fonic acid (DIDS) is another type of anion channel blocker, which 5 s -10 -5 051015 (min) has been shown to block glutamate release from astrocytes (28). DIDS (1 mM) abolished the glia-photostimulated PC current (P < 2+ TTX,Cd ,PIC +DIDS 1.5 0.001, n = 6; Fig. 2C, Upper), whereas the BG photocurrent Blue 1 remained intact (P > 0.05, n = 5; Fig. 2C, Lower). No change in the Yellow 0.5 current responses of PCs to exogenous puff application of glutamate was observed in the presence of DIDS (Fig. S7C). These BG 0 Norm. amp.

results suggest that glutamate release by glia photostimulation is NEUROSCIENCE 200 pA -10 -5 051015 likely via anion channels not sensitive to NPPB but sensitive to 5 s (min) DIDS (29). Fig. 2. Glia photostimulation triggers glutamate release. Glia-photostimulated – PC currents (Left) and time course of the amplitude change (Right) before and Glial Activation Induces Long-Term Depression of PF PC Synapses. As after bath application of drugs (drug application from time = 0). Amplitudes BG processes are closely situated to the PFs in the molecular layer of the current are plotted against time with the amplitude normalized to (ML), it is possible that glia photostimulation induces activation of 2+ account for the rundown as described in Fig. S5C.(A) The PC current was the PFs. In the absence of TTX and Cd , such interaction could completely eliminated by GYKI53655 (n = 5; P < 0.001 0–2 min before versus lead to neuronal glutamate release from PF synapses, which could 10–14 min after drug application, paired t test). (B) Inhibition of glutamate lead to the amplification of the glia-photostimulated PC current. To transporter by TBOA augmented the PC current (n = 5). (C) Inhibition of anion examine this possibility, photostimulation was restricted to the ML channels with DIDS abolished the PC current (Upper; n = 6, P < 0.001), whereas to selectively activate the BG processes (Fig. 3A, Left). The PC the BG photocurrent per se was not significantly altered by DIDS (Lower; n = 6, currents produced by the ML photostimulation were unaffected by P > 0.05). TTX application (P > 0.05, n = 5; Fig. 3A, Upper). This result indicates that the activation of BG processes alone is sufficient to trigger glia-photostimulated PC current and it is unlikely that neu- μ threo β 100 M DL- - -benzyloxyaspartic acid (TBOA), glutamate ronal glutamate release is triggered. In contrast, photostimulation fi transporter blocker, caused a signi cant increase in the amplitude of the region including both ML and GL evoked PC current, which n = B of the glia-photostimulated PC current ( 5; Fig. 2 ). Glutamate was significantly reduced by TTX treatment (P < 0.01, n = 5; Fig. transporter blockade likely increased the effective concentration of 3A, Lower). Because weak EYFP (Fig. 1A)andtTA (Fig. S2B) glutamate reaching the AMPARs. Although BG-AMPARs are signals were detected in the GL, protoplasmic astrocytes in the GL known to rapidly desensitize (6), in the presence of CTZ and could also be photostimulated as well as the BGs, although the TBOA, the released glutamate acting onto BG-AMPARs could be EYFP signal per unit area was 3.3-fold higher in the ML compared detected (Fig. S4F). Taken together, these lines of experiments with the GL. This result suggests that wide-field photostimulation demonstrate that glia photostimulation causes releases of glu- not only causes glutamate release from BGs but also causes GC tamate, and the released glutamate reaches the low-affinity PC firing via activation of GL astrocytes in the absence of TTX and = μ fi 2+ AMPARs [EC50 432 M(24)]atasuf cient concentration. Cd . This result accounts for the observation that not only PCs The above pathway is the straightforward explanation of our fos + but also GCs were positive for c- after the photostimulation in results; however, as K is also likely to be released upon glia vivo (Fig. 1E). photostimulation (Fig. S3 C and D), alternative pathways leading The released glutamate can activate not only AMPARs but also from glia photostimulation to PC current could be speculated. + metabotropic glutamate receptors type 1 (mGluR1), which are First, it is possible that [K ]o increase causes depolarization of PCs expressed on the peripheral of the PC spines and dendrites (30), and results in release of glutamate from PCs (25), even though the + but have not been detected on cerebellar glial cell membrane (31). recordings were made in the presence of TTX and Cd2 . Direct Activation of mGluR1 is essential for inducing synaptic plasticity depolarization of PCs can be accomplished by ChR2-mediated at PF–PC synapses and for coordinated motor behavior (32). BG excitation using PV-tTA::tetO-ChR2(C128S) mice (Fig. S6A). processes are situated immediately adjacent to the mGluR1 on Although multiple neighboring PCs were likely to be depolarized PCs, which raises a possibility that glutamate released from glia by PC photostimulation, currents evoked in the recorded PC were photostimulation could induce changes in neurotransmission from unaffected by NBQX, suggesting that depolarization of neigh- PFs to PCs. To test this, we examined the effect of the glia pho- boring PCs does not cause glutamate release detectable by PC tostimulation on PF–PC excitatory postsynaptic currents (EPSCs) AMPARs. Second, it is possible that glia is releasing glutamate but (Fig. 3B). A single 10-s photostimulation to the ML+GL region the released glutamate acts on neighboring cells other than the induced long-term depression (LTD) of PF–PC EPSCs (P < 0.05, + PCs and the depolarization of these neighboring cells causes [K ]o n = 5; Fig. 3 C and D). Application of an mGluR1 antagonist,

Sasaki et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 150 restricted to the ML. As shown above, neuronal glutamate release A ML stim Blue Yellow from PFs was not likely to be induced by this photostimulation (Fig. 100 3A). LTD was induced to a similar level as with the ML+GL +TTX photostimulation (Fig. 3D). This suggests that glutamate release 50 from glia was the underlying mechanism of glia-photostimulated patch PC LTD. However, it still remained possible that glia photostimulation Amplitude (pA) photo 0 caused depolarization of the recorded and the surrounding PCs, 30 pA +TTXPIC 5 s which could somehow lead to LTD. To test this possibility, direct depolarization of PC was induced by using PV-tTA::tetO-ChR2 ML+GL stim Blue 600 (C128S) mice. No LTD was triggered by the selective PC photo- Yellow stimulation (Fig. S8B). The above results demonstrate that glial 400 release of glutamate not only can activate PC AMPARs but also +TTX can activate mGluR1 and initiate pathways leading to LTD. 200 patch PC Amplitude (pA) Brief Photostimulation Triggered Glutamate Release. Photostimulation photo 400 pA 0 +TTXPIC of BGs for 10 s resulted in the depolarization of the BGs by 10.0 ± 5 s 0.7 mV (Fig. S9A), which was comparable to that induced by strong electrical stimulation in the MLs (100 V × 20; Fig. S9D). Using B PF-PC EPSC more moderate stimulation (60 V × 10), typically used in acute before 0-5 min after 20-25 min slice experiment for studying burst activity of PF–PC synapses, BGs ML+GL stim were depolarized by 3.6 ± 0.5 mV (n = 6; Fig. 4A). Photo- Blue stimulation with short light pulses (200-ms blue and 200-ms yellow with an interval of 500 ms) evoked comparable depolarization of Yellow the BGs by 2.7 ± 0.5 mV (n = 6; Fig. 4A). This same light pattern was used in the in vivo experiments (Fig. 1D). PC 200 pA 100 pA To evaluate the minimum amount of glia photostimulation 5 s 50 ms required for detecting glutamate release, CTZ and TBOA were coapplied to maximally increase the sensitivity of PC AMPARs CDE to glutamate. In this condition, a 100-ms blue pulse was sufﬁcient B 1 ** 2 to evoke detectable current response in PCs (Fig. 4 ). Such short light pulse evoked depolarization of BGs by 1.4 ± 0.2 mV (Fig. 1 A 0.5 1 S9 ). This depolarization was comparable to the depolarization evoked by paired-pulse electrical stimulation of PFs (60 V×2; Fig. S9D). This implies that such weak stimuli are sufﬁcient to Norm. amp. 0 0 0.5 evoke glutamate release from glia. The amplitude of the glia-

after after photostimulated PC current increased proportionally with the Paired-pulse ratio Norm. amp. ML+GL stim before before duration of blue light application (Fig. 4C). ML stim

LY367385 LY367385

0 ML+GL stim Glia Activation Drives Behavioral Changes. We next aimed to eval- -10 0210 030 uate whether signals initiated from the glia could sufﬁciently (min) LY367385

ML+GL stim propagate through the neuronal network and ultimately drive behavioral responses in vivo. For this purpose, we used the moderate Fig. 3. Synaptic plasticity induced by glia photostimulation. (A)(Upper) fi fi light sequence that produced depolarization of BGs to comparable When photostimulation was aimed speci cally to the ML, no signi cant levels as with electrical PF burst stimulation (Fig. 4A). Photo- change in the amplitudes of glia-photostimulated PC current was observed stimulation was applied through optical fibers inserted close to the after application of TTX (n = 5 cells; P > 0.05, paired t test). (Lower)PC fl A fos currents evoked by photostimulation of a region including both ML and GL cerebellar occulus region (Fig. 5 ), and an induction of c- fi = < mRNA in a subset of PCs, GCs, and SC/BCs was observed by the were signi cantly reduced by TTX (n 5 cells; P 0.001, paired t test). EYFP B was bleached after the recording to confirm the location of photo- glia photostimulation (Fig. 5 ). According to slice experiments, the – mechanisms underlying the glia-photostimulated neural activity are stimulation. (B) Electrically stimulated PF PC EPSCs in response to paired- + pulse protocol (100-ms interval) before and after a single ML+GL photo- likely due to K efflux and glutamate release. In this condition, we stimulation. LTD of the PF–PC EPSC was observed. (C) Time course of the first assessed the effect of glia photostimulation on the eye movement PF–PC EPSC amplitude (ML+GL stim, n = 5; LY367385, n = 5; *P < 0.05, (Fig. 5C). Amplitude increase of the horizontal optokinetic reflex Student t test). Photostimulation was applied at time 0. (D and E) Summary (HOKR) upon repetitive visual stimuli has been shown to be of the changes in the normalized EPSC amplitude (D) and the mean paired- controlled by the activity of the flocculus (33, 34). Glia photo- pulse ratio (E) before and 20–25 min after the glia photostimulation. Pho- stimulation was done while this HOKR was induced and pertur- tostimulation of ML alone was also sufficient to produce LTD (n = 7). All bation of the smooth eye pursuit of the visual stimuli was evoked recordings were done in the presence of PIC. nearly every time the photostimulation was applied (Fig. 5D, Movie S1). In addition, pupil dilation was also evoked (Fig. 5 E and F), which is in agreement with the fact that the cerebellum and the μ P > n = D 100 M LY367385, blocked the LTD ( 0.05, 5; Fig. 3 ). No deep cerebellar nuclei modulate pupil size (35). After the pupil change in the input resistance of PCs was detected after photo- area came back close to the basal value (Fig. 5F), the HOKR stimulation (before, 100.6 ± 10.5 MΩ; after, 105.6 ± 8.9 MΩ; P > amplitude was measured and an increase compared with the 0.05, paired t test). The photostimulation did not significantly alter baseline was observed (Fig. 5 E and G). This effect lasted for the EPSC paired-pulse ratio (P > 0.05, n = 5; Fig. 3E) nor the a couple of minutes. Despite the perturbation of eye movement as number of fibers activated by the electrical stimulation, as exam- well as the transient increase in the HOKR amplitude, HOKR ined by fiber volley recordings (Fig. S8A), implying that pre- amplitude increase after 1-h continuous visual training was ob- synaptic components were not affected by the photostimulation. served in most mice as in the ChR2(−)mice(Fig. S10). As we have shown that wide-field ML+GL photostimulation likely activates GCs via activation of GL astrocytes, it is possible that GC Discussion firing subsequent to glia photostimulation alone was sufficient to Using optogenetics, we showed that selective glia photostimulation induce LTD. To exclude this possibility, photostimulation was drives neuronal activity, synaptic plasticity, and behavioral response,

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1213458109 Sasaki et al. Downloaded by guest on September 28, 2021 ﬁ A E-stim (60V x10) Photostim 4 also showed that glia photostimulation for 100 ms was suf cient to Blue trigger depolarization of BGs (Fig. S9A) and release of glutamate Yellow 2 was detectable with PC AMPARs (Fig. 4C). Such depolarization

2 mV 0 BG 2 s Amplitude (mV) midline Light A 2.7 mm B No light E-stim c-fos B C Photostim +CTZ,TBOA 1.5 mm 100 ms 500 ms 1000 ms +CTZ,TBOA Blue 1 Yellow 1 mm

Norm. amp. flocculus PC 0.1 C 100 pA 100 pA 200 pA 10 100 1000 10000 1 s 1 s 1 s (ms)

Fig. 4. Brief photostimulation was sufficient in inducing glutamate release. (A) A typical electrical stimulation used in acute slice experiments depolarized BGs. Short photostimulation (200-ms blue and 200-ms yellow light with an interval of 500 ms in the absence of any drugs) produced similar depolarization. (B) Glia-photostimulated PC currents in response to various durations of blue and fixed duration (200 ms) of yellow light pulses. Glutamate release was detected with pulses of blue light as short as 100 ms. These recordings were made under the condition that detection sensitivity of AMPARs on PCs was D + * maximally amplified by coapplication of CTZ and TBOA (plus TTX, Cd2 ). (C) Summary of the amplitudes of the PC currents plotted as a function of duration Horizontal eye position 100

of blue light (control, ﬁlled circles, n = 5; CTZ and TBOA, open circles, n = 5). All NEUROSCIENCE recordings were done in the presence of PIC.

50 and that the glia-to-neuron signaling is mediated by glial release of glutamate through DIDS-sensitive anion channels. 0 ChR2 (-) ChR2 (+)

20 deg Deviation from mean (deg·s) Mechanism Underlying Glia-Driven Neuronal Activity. Rapid glia-to- 5 s neuron communication could be mediated by extracellular gluta- + E F mate and/or K (36). Glia photostimulation likely caused changes + + in extracellular K ,asK channel-mediated currents in addition to Pre 300 40 ChR2-mediated currents were observed in BGs (Fig. S3D). In 200 + 100% 20 addition to the accumulation of extracellular K , we suggest PCs Pupil area 100 60 s are excited by glutamate released from glia photostimulation. 0 0 Evidence supporting AMPAR and mGluR1 activation following Post Pupil area (%) Max Recovery time (s) glia photostimulation was shown (Figs. 2 and 3, and Fig. S4)and ChR2 (-) the AMPAR-mediated PC currents were blocked by DIDS, which G * ChR2 (+) Horizontal eye position blocks transporters and channels that allow the movement of 100 After negatively charged molecules, including glutamate. We propose Before that these DIDS-sensitive channels not only control the ambient 50 glutamate level (29) but also regulate the amount of glutamate 5 deg 0 released in response to rapid and transient activity of glia. Glial Before 0-2 min 2-4 min

1 s Relative gain (%) depolarization alone or movement of ions associated with ChR2 (C128S) likely triggers glutamate release through the DIDS-sensitive Fig. 5. Glia-driven behavior. (A) Schematic of the optical fiber insertion location anion channels; however, the mechanism leading from photo- in the brain atlas (6.0 mm posterior to the bregma). (B) Expression of c-fos mRNA activation to the glutamate release need to be evaluated further. was detected in the PC layer and ML/GL after a single incident of photostimulation An important issue is whether the same glutamate release mech- (200-ms blue and 200-ms yellow light with an interval of 500 ms) delivered through anisms operate under physiological conditions. In our experi- the optical fibers. (C) A photo of the HOKR experiment apparatus. (D)HOKRwas ments, 10-s glia photostimulation resulted in depolarization of up inducedinhead-fixed unanesthetized mice. The absolute difference was taken to 10 mV in BGs (Fig. S9A). Glia photostimulation likely induces between the average horizontal eye movement (gray; 5 min before glia photo- depolarization homogeneously throughout the cells, as ChR2 is stimulation) and the eye movement after glia photostimulation (black; same expressed throughout the entire processes. When PFs were elec- durations as above) via optic fibers inserted into the cerebellum and was in- trically stimulated repetitive times, BGs responded with a compa- tegrated for 25 s [n = 6 and 5 animals for ChR2(−) and ChR2(+), respectively; *P < rable amplitude of depolarization (Fig. S9 B–D). However, 0.01, Student t test]. (E)(Middle Right) Pupil area plotted against time. (Bottom because of the low input resistance, much of the depolarization Right) Two-minute average of the horizontal eye movement before the glia photostimulation (gray) and after the pupil area came back within 1 SD from the produced locally is likely to be attenuated and underestimated. A baseline (black). (F) Peak increase in the pupil area and time for the cessation of computational model has estimated that depolarization as much pupil dilation were summarized (n = 5 animals). The time it took for the pupil to as 40 mV is likely produced at the ensheathing BG membrane in return within 1 SD from the baseline was defined as the recovery time. (G)The response to synaptic stimuli (7); therefore, similar or even larger average gain of horizontal eye movement at 0–2minand2–4 min after cessation depolarization compared with our BG photostimulation protocol of pupil dilation were normalized to the gain before the glia photostimulation for could be produced physiologically, although such a profound de- ChR2(+) mice. As pupil dilation did not occur in ChR2(−) mice, eye movements were polarization has not been substantiated experimentally due to measured after 0–2minand2–4 min after 45 s from photostimulation. Results inaccessibility of glial fine processes by recording electrodes. We were summarized for ChR2(−) and ChR2(+)(n = 6and5;*P < 0.05, Student t test).

Sasaki et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 of BGs can also be evoked by a couple of electrical stimulation 12–15). In this study, we directly demonstrate that signals initiated typically used for studying neuronal synaptic transmission (Fig. S9 from glial cells can drive neuronal activity and animal behavior. B–D); therefore, glial release of glutamate may have unintentionally We expect that our model can be a useful tool to assess how the been evoked in previous studies, which could complicate the in- “debate” between neurons and glia can set the tone of our mind. terpretation of those studies. Materials and Methods Plasticity Induced by Glia Photostimulation. We demonstrated that Full details of all methods are available in SI Materials and Methods. All animal glia photostimulation could trigger long-term plasticity of PF–PC procedures were conducted in accordance with the National Institutes of synaptic transmission. In a previous report, stimulation of sparsely Health Guide for the Care and Use of Laboratory Animals (38) and approved by distributed PFs failed to evoke LTD, whereas stimulation of PF the Animal Research Committee of the National Institute for Physiological bundles in the ML readily evoked LTD (37). The latter stimulation Sciences. For slice electrophysiology, parasagittal cerebellar slices were pre- protocol inevitably activates the BGs as well (9, 10). We did not pared from young mice [postnatal day 17 (P17) to P24]. For in vivo optical fi evaluate whether glial release of glutamate alone is sufficient in stimulation, blue and yellow light were applied through a plastic optical ber producing LTD with weaker glia photostimulation than the 10-s (0.75 or 0.5 mm in diameter) to the cerebellum. For HOKR experiment, a sheet of paper with checkered pattern was placed semicircularly (radius, 32 cm) protocol. Because mGluR1 is expressed at a location that is surrounding the mouse and oscillated horizontally and sinusoidally. heavily protected by glutamate transporters, summation of glutamate concentration from neuronal spillover and glial release may ACKNOWLEDGMENTS. We thank A. Yamanaka for helping with the optimi- be required to overcome the protection and activate the mGluR1. zation of the tetO-ChR2(C128S)-EYFP transgenic mice, T. Tsunematsu for Glial glutamate release could thus be considered as an amplifi- assisting in the setting up for the general in vivo experiments, K. Ikenaka and cation mechanism for detection of close by activity and induction S. Sugio for helping with the in situ hybridization and immunohistochemistry of plasticity. However, it still remains unresolved whether our experiments, and T. Sakatani, W. Wen, and W. Aziz for setting up the HOKR apparatus. This work was supported by grants from Grant-in-Aid for Scientific stimulation protocol was physiologically relevant. Further studies Research on Innovative Areas “Mesoscopic Neurocircuitry” from the Ministry are required to reveal participation of glial cells in long-term of Education, Culture, Sports, Science and Technology of Japan (MEXT) synaptic plasticity under physiological conditions. (23115521) (to K.M.), Grant-in-Aid for Scientific Research (C) from MEXT [22500362 (to K.M.) and 21500311 (to Y.F.)], Precursory Research for Embry- Conclusion and Future Perspective. We generally assume that our onic Science and Technology from Japan Science and Technology Agency (JST) (to K.M.), Core Research for Evolutional Science and Technology from JST (to mind is dictated solely by the activity of neurons. Contrary to this Y.F.), Solution-Oriented Research for Science and Technology from JST (to prevailing notion, recent evidence proposed a potential role of glia R.S.), Grant-in-Aid for Young Scientists (A) from MEXT (23680042) (to K.F.T.), in actively participating in the neural information processing (1, and the Takeda Science Foundation (to K.F.T.).

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