Astrocytes respond to neuronal activity and were shown to be necessary for plasticity and memory. To test whether astrocytic activity is also sufficient to generate synaptic potentiation and enhance memory, we expressed the Gq-coupled receptor hM3Dq in CA1 astrocytes, allowing their activation by a designer drug. We discovered that astrocytic activation is not only necessary for synaptic plasticity, but also sufficient to induce NMDA-dependent de novo long-term potentiation in the hippocampus that persisted after astrocytic activation ceased. In vivo, astrocytic activation enhanced memory allocation; i.e., it increased neuronal activity in a task-specific way only when coupled with learning, but not in home-caged mice. Furthermore, astrocytic activation using either a chemogenetic or an optogenetic tool during acquisition resulted in memory recall enhancement on the following day. Conversely, directly increasing neuronal activity resulted in dramatic memory impairment. Our findings that astrocytes induce plasticity and enhance memory may have important clinical implications for cognitive augmentation treatments.

To explore the role of astrocytes in synaptic activity and plasticity, as well as in memory performance, we employed chemogenetic and optogenetic tools in this cell population and found that astrocytic activation resulted in increased spontaneous vesicle release, and de novo synaptic potentiation mediated by NMDA. These plastic changes resulted in enhanced memory allocation and improved cognitive performance, which could not be directly achieved by elevating neuronal activity.

The studies above elegantly show that astrocytes are necessary for long-term plasticity and normal memory performance, but it is unknown whether astrocytic activity is not only necessary but also sufficient to induce synaptic plasticity and enhance cognitive performance. Beyond the clinical implications of memory enhancement, such an investigation could illuminate the complex way in which astrocytes do not merely respond to the neighboring neural network activity and support it, but rather precisely modulate the way it processes information.

Although the supportive roles of astrocytes are well recognized, their direct effects on neuronal activity remain elusive. Pioneering studies have examined how astrocytes monitor and directly modulate neuronal activity, and support the idea of a “tripartite synapse,” in which astrocytes do not merely encapsulate and insulate synapses, but also sense and actively modify synaptic activity (). Most of these studies were conducted on a single cell level, for example, by patching a single astrocyte to modulate its activity () or by uncaging Cain single astrocytes (). These techniques cannot be used in behaving animals, where modulating the activity of a population of astrocytes is required (). Because of this difficulty, only a handful of studies have directly investigated the necessity of astrocytes in mammalian normal memory ().

Release of gliotransmitters through astroglial connexin 43 hemichannels is necessary for fear memory consolidation in the basolateral amygdala.

Memory stands at the heart of cognitive function, guiding future behavior based on past experience. Memory disruption is relatively easy to induce, whereas memory enhancement has challenged scientists for many years. The majority of cognitive enhancement models involve alterations in synaptic function, often via direct or indirect effects on NMDA-R signaling (). Another way to improve memory is to boost the process of memory allocation, the selection of the neuronal ensemble that will serve as the physical basis underlying the specific memory ().

Classical opsins, directly affecting the membrane potential, were used in the past to manipulate astrocytes in behaving mice (), though never in the hippocampus or in the cognitive tasks. It is not yet fully understood how such changes in membrane potential, which do not mimic any physiological processes, affect astrocytic function. Thus, we used the Opto-α1AR opsin, a light-sensitive Gq-coupled receptor (), which is termed “OptoGq,” to manipulate the Gq pathway in astrocytes in real time. This opsin was shown to specifically recruit the inositol triphosphate (IP) pathway and induce the release of Cafrom intracellular storage in HEK cells and in astrocytes (). We have produced an AAV viral vector to express OptoGq fused to eYFP under the GFAP promoter (AAV1-GFAP::OptoGq-eYFP) and delivered it to the CA1. OptoGq expression was limited to the astrocytic outer membrane ( Figure 7 A), with high penetrance (>87% of the GFAP cells expressed OptoGq) ( Figure 1 B) and almost complete specificity (>98% OptoGq-positive cells were also GFAP positive) ( Figure 1 C). To verify that optogenetic astrocytic activation does not have a direct effect on exploratory behavior that may alter freezing, we tested GFAP::OptoGq mice in an open field and found no effect of light administration (473 nm, 20 Hz, 45-ms pulse duration) on exploration ( Figure 7 D). To specifically test the effect of astrocytic activation on fear memory acquisition, we administered light only during the 5-min training session. No real-time effect of light on immediate freezing following shock administration was observed ( Figure 7 E). Remarkably, light administration during training resulted in 89% elevation in contextual freezing in GFAP::OptoGq mice tested a day later ( Figure 7 E) (p < 0.05, t test). No effect on auditory-cued memory in a novel context was observed, i.e., both groups demonstrated similar freezing in response to the tone ( Figure 7 F) (F= 11.19, time main effect p < 0.01). To conclude, we show that real-time optogenetic astrocytic activation only during conditioning enhances memory acquisition, as demonstrated by improved recall a day later.

(F) Optogenetic activation of CA1 astrocytes during training had no effect on the acquisition of the hippocampal-independent auditory-cued fear memory in OptGq mice compared to controls, with both groups showing increased freezing during tone presentation ( ∗ p < 0.01). Data are presented as mean ± SEM.

(E) Optogenetic activation of CA1 astrocytes during fear-conditioning training had no effect on immediate freezing at the time of illumination, but increased contextual freezing by 89% ( ∗ p < 0.05) on the next day in GFAP::OptoGq (n = 5) mice compared to eYFP (n = 5) controls.

(D) Optogenetic activation of CA1 astrocytes had no effect on exploration of a novel environment, because eYFP control (n = 5) and GFAP-OptoGq (n = 5) mice explored the field with similar path lengths before, during, and after light administration.

(B and C) GFAP::OptoGq was expressed in 87.4% of CA1 astrocytes (58/65 cells from 2 mice) (B), with 98.7% specificity (57/58 cells, from 2 mice) (C).

(A) Bilateral double injection of AAV1-GFAP::OptoGq-eYFP resulted in OptoGq expression in CA1 astrocytes only: OptoGq (green) was expressed in the astrocytic membrane around the soma and in the distal processes (scale bar, 50 μm).

Our results show that chemogenetic astrocytic activation improves memory only when induced during acquisition, but not during recall. However, because CNO was administered 30 min before the task and remained in the body for several hours afterward, it could have affected not only memory acquisition but also early consolidation. To demonstrate the involvement of astrocytes specifically at the acquisition stage, we employed optogenetics, providing both cell-type specificity by confining expression to astrocytes and a strict temporal control by light administration, making it highly suitable for memory research ().

Our results show that astrocytic activation enhances neuronal activity in a task-dependent way, as demonstrated by its lack of effect on activity in home-caged mice, as opposed to the astrocyte-induced elevation in neuronal recruitment during memory allocation. Directly activating neurons, on the other hand, causes a non-selective increase in activity regardless of external experiences. This could explain why chemogenetic astrocytic activation improves memory, whereas neuronal activation impairs it.

Thus, we sought to test whether astrocyte-induced memory enhancement is not merely due to the general increase in neuronal activity, but rather stems from a tailored response of astrocytes to the activity of their surrounding neurons. To achieve that, we activated astrocytes or neurons in vivo, either in home-caged mice or in mice that acquired FC, and then measured cFos levels in CA1 neurons, as a marker for neuronal activity. Mice were bilaterally injected with AAV8-GFAP::hM3Dq-mCherry or AAV8-CaMKII::hM3Dq-mCherry or AAV8-GFAP::eGFP to the dorsal CA1, and 3 weeks later CNO (3 mg/kg, i.p.) was administered in the home-cage or 30 min before FC. Brains were collected 90 min later and stained for cFos ( Figure 6 A). FC increased cFos levels in all saline-injected mice ( Figures 6 B and 6C) (p < 0.05, t test). CNO administration to GFAP::hM3Dq mice in vivo increased neuronal activity beyond the threshold for cFos expression compared to saline-injected mice only when coupled with learning, but not in home-caged mice ( Figures 6 B and 6D) (p < 0.05, t test). CNO administration in CaMKII::hM3Dq mice increased neuronal cFos levels regardless of training, in both fear conditioned and home-caged mice ( Figures 6 C and 6E; p < 0.05 for both, t test). CNO alone had no effect on cFos levels in GFAP::eGFP mice in any condition ( Figures S7 A–S7C).

(C) Representative cFos expression images are shown (GFAP-eGFP in green, nuclei in blue, cFos in white; Scale bar 100μm). Data presented as mean ± SEM.

(B) CNO application had no effect on neuronal activity, as measured by cFos expression, regardless of memory acquisition.

CNO Application by Itself Has No Effect on Neuronal Activity, Related to Figure 6

(D and E) Representative images from mice expressing hM3Dq-mCherry (red) in their CA1 astrocytes (D) or neurons (E) that were injected with saline or CNO. Brain slices were stained for c-Fos (green) and the nuclear DAPI stain (blue) following either home-cage exposure or memory acquisition. cFos-expressing, activated astrocytes whose nuclei are out of the CA1 neuronal cell layers are clearly visible only in the GFAP-hM3Dq CNO-injected groups. Image frame colors correspond to group bars colors. Scale bar, 100 μm. Data are presented as mean ± SEM.

(C) CNO administration increased neuronal cFos expression in CaMKII-hM3Dq mice regardless of external input in both home-caged and fear-conditioned mice ( ∗ p < 0.05 for both).

(B) CNO administration had no effect on neuronal cFos expression in GFAP::hM3Dq home-caged mice, but increased neuronal activity only when it was combined with fear memory acquisition ( ∗ p < 0.05).

(A) CNO was administered to mice injected with AAV8-GFAP-hM3Dq-mCherry or AAV8-CaMKII-hM3Dq. 30 min later, mice either underwent fear-conditioning acquisition or remained in their home cages. 90 min after that, brains were removed, sliced, and stained for cFos (n = 4–9 mice, 16–34 slices per group).

Our results show that astrocyte-mediated potentiation of neuronal activity enhances memory whereas direct neuronal activation dramatically impairs it. Previous research had demonstrated that a small number of neurons active before training are more likely to be allocated to the engram supporting an acquired memory (), that such ensembles in CA1 are later necessary for recall () and that increasing the activity of a small neuronal population in the BLA before FC acquisition can improve fear memory ().

Here, we show that as opposed to astrocytic activation, which induces LTP and enhances FC performance, a direct chemogenetic stimulation of CA1 neurons dramatically impairs contextual memory.

We then replicated these results, this time manipulating a larger population of neurons, but activating them mildly, using a lower CNO dose (0.5 mg/kg) ( Figure S6 A). Again, no effects on context exploration before conditioning or immediate freezing following shock administration were observed ( Figures S6 B and S6C). Furthermore, neuronal activation with these new parameters during training again resulted in reduced contextual freezing a day later ( Figure S6 C) (p < 0.05, t test), and had no effect on auditory-cued memory in a novel context, as both groups demonstrated similar freezing in response to the tone ( Figure S6 D) (F= 133.276, time main effect p < 0.00001).

(D) Neuronal activation in CA1 had no effect on auditory-cued freezing in a novel context, with both groups showing increased freezing during tone presentation (p < 0.00001). Data presented as mean ± SEM.

(C) CNO application before training induced a 55% decrease in contextual freezing in CNO-treated mice tested 24 hours later, compared to saline treated controls (p < 0.05).

(A) hM3Dq was expressed in CA1 neurons (Scale bar 100μm). Mice expressing hM3Dq in their CA1 neurons were injected with either saline (n = 6) or a low dose of CNO (0.5mg/kg; n = 6) 30min before fear conditioning acquisition.

Our findings that astrocytic activation resulted in increased synaptic transmission, de novo plasticity and improved memory, raises the tempting hypothesis that astrocytes react to neuronal activity around them and modulate it in a physiologically meaningful way, leading to improved coding of contextual information. However, we cannot exclude the possibility that the observed memory enhancement could have been caused by a general increase in hippocampal neuronal activity induced by the manipulated astrocytes. To test whether directly increasing neuronal activity results in similar effects, we sought to stimulate CA1 neurons and test the consequent changes in cognitive performance. To that end, we injected mice with an AAV8 vector encoding hM3Dq-mCherry under the control of the CaMKIIα promoter, specific to glutamatergic neurons. hM3Dq was shown in the past to increase firing, elevate fEPSP size and enhance LTP in CA1 pyramidal neurons, but it also poses a risk of seizures (). To avoid this risk, we aimed for a moderate expression level. Stereotactic delivery of the AAV8-CaMKIIα::hM3Dq-mCherry vector resulted in CA1-specific expression ( Figure 5 A). hM3Dq was exclusively expressed in neurons ( Figure 5 B) with moderate penetrance (47% of the NeuN cells expressed hM3Dq) and complete specificity (>98% hM3Dq-positive cells were also NeuN positive) ( Figure 5 C). Co-staining with the astrocytic marker GFAP or the microglial marker Iba1 showed no overlap with hM3Dq expression ( Figures 5 D and 5E). To test the effect of direct neuronal activation on memory acquisition, we injected CaMKIIα::hM3Dq mice with CNO (3 mg/kg, i.p.) 30 min before FC acquisition. First, we verified that this manipulation does not induce seizures or affect motor function, by quantifying the exploration of the conditioning cage during the 120 s before the introduction of the first tone. No difference between hM3Dq-mCherry mice treated with saline or CNO was observed ( Figure 5 F). Mice were then FC and tested on the next day. Interestingly, neuronal activation during training resulted in dramatically reduced, rather than improved, contextual freezing one day later ( Figure 5 G) (p < 0.01, t test). No significant effect on auditory-cued memory in a novel context was observed, as both groups demonstrated similar freezing in response to the tone ( Figure 5 H) (F= 48.165, time main effect p < 0.00001).

(H) Neuronal activation in CA1 had no effect on auditory-cued freezing in a novel context, with both groups showing increased freezing during tone presentation ( ∗ p < 0.00001). Data are presented as mean ± SEM.

(D and E) No co-localization with the astrocytic marker GFAP (D) or the microglial marker Iba1 (E) was detected (scale bar, 50 μm). Mice expressing hM3Dq in their CA1 neurons were injected with either saline (n = 9) or CNO (n = 8) 30 min before fear conditioning acquisition.

(C) hM3Dq was expressed in 47% (442/913 cells, from two mice) of CA1 neurons, with >98% specificity (442/446 cells, from two mice).

(B) hM3Dq was expressed in the neuronal membrane around the soma and in the apical and the basal dendrites of CA1 neurons (scale bar, 30 μm).

(A) Bilateral double injection of AAV8-CaMKII::hM3Dq-mCherry resulted in relatively sparse hM3Dq expression in CA1 only (scale bar, top, 300 μm, and bottom, 100 μm).

Our behavioral results show that astrocytic activation during memory acquisition and early consolidation is sufficient to improve memory retrieval in two cognitive tasks.

To verify that astrocytic activation does not have a direct effect on exploratory behavior or anxiety, which may result in increased freezing, we tested free exploration of an open field in the same GFAP::hM3Dq mice. CNO application had no significant effect on either total exploration or anxiety-related behavior, measured as the percent time spent in the central 35% of the arena ( Figure 4 J). To confirm that our results did not stem from the CNO application itself, we trained additional cohorts of mice, injected with a control AAV8-GFAP::eGFP vector ( Figure S5 A) in the same repertoire of behavioral paradigms. CNO application in these control mice had no effect on any behavior ( Figures S5 B–S5K).

(J and K) When administered during recall only, CNO administration to eGFP-expressing mice (n = 4) had no effect on contextual or auditory-cued memory compared to saline injected mice (n = 4). Data presented as mean ± SEM.

(I) CNO administration during recall on the next day also had no effect on retrieval.

(F–H) CNO administration before training to eGFP-expressing mice had no effect on exploration of the conditioning chamber (F) contextual memory (G) or auditory cued memory (H) one day later.

(C and D) Saline-treated (n = 7) and CNO-treated (n = 7) eGFP mice explored the T-maze with similar path lengths (C) and there was no effect on new arm recognition (D). (E) Representative exploration traces. Mice expressing eGFP in their CA1 astrocytes were injected with either saline (n = 7) or CNO (n = 7) 30min before fear conditioning acquisition.

(B) In a novel environment, saline-treated (n = 7) and CNO-treated (n = 7) eGFP mice explored the field with similar path lengths (top) and there was no effect on anxiety (bottom), as the percent of time that saline and CNO treated mice spent in the center of the open field was similar. Representative exploration traces are presented.

To further differentiate between the roles of astrocytes during acquisition and recall, we injected two new cohorts of mice with AAV8-GFAP::hM3Dq-mCherry to their CA1, and administered CNO 30 min before FC acquisition in one cohort, and 30 min before recall in the other. Importantly, in the first new cohort, we replicated our initial observation that astrocytic activation during acquisition enhanced recall on the next day ( Figure S4 E) (p < 0.005, t test), without affecting auditory-cued memory ( Figure S4 F) (F= 47.57, time main effect, p < 0.00001), or exploration of the chamber before conditioning ( Figure S4 D). In the second cohort, CNO was not administered during acquisition, but rather 30 min before the recall test. GFAP::hM3Dq mice injected with CNO at that time showed similar contextual ( Figure 4 H) and auditory-cued ( Figure 4 I) freezing, compared to saline controls. These findings suggest that astrocytes confer their cognition-enhancing effects during memory acquisition, and possibly early consolidation, but not during memory recall.

To better define the memory stage affected by astrocytes, and extend our findings to an additional cognitive task, we administered CNO 30 min before FC training, pairing a foot-shock with a novel context and an auditory cue, in a new cohort of mice. No effect of CNO on exploration of the context before conditioning ( Figure S4 C) or immediate freezing following shock administration ( Figure 4 E) was observed. One day later, mice were placed back in the conditioning context, and freezing was measured. Remarkably, CNO application during training resulted in a 40% elevation in contextual freezing in GFAP::hM3Dq mice tested 24 hr after acquisition, when CNO was no longer present () ( Figure 4 E; p < 0.015, t test). To establish the spatial specificity of our manipulation, we verified that the effect of CA1 astrocytic activation is unique to the hippocampal-dependent contextual memory task: Indeed, no effect was observed when the same mice were tested for auditory-cued memory in a novel context, i.e., both groups demonstrated similar freezing in response to the tone ( Figure 4 F) (F= 106.04, time main effect, p < 0.00001). CNO application during recall in the conditioning context on the next day did not further alter recall, but the original improvement was still evident ( Figure 4 G).

To test the effect of astrocytic activation on cognitive performance, CNO was administered 30 min before T-maze training, in which mice were exposed to 2 arms of the maze for 20 min, and 5 min later were re-introduced to the maze, with all three arms now available for exploration. CNO application resulted in a significant elevation in novel arm preference compared to saline-injected controls ( Figures 4 C and 4D) (p < 0.05, t test). No effect of CNO on overall maze exploration was observed ( Figure S4 B). In this experiment, astrocytes were activated during both acquisition and recall, and could have contributed to cognitive enhancement at either stage.

Based on our finding that astrocytic activation is sufficient to induce neuronal potentiation, and on previous research demonstrating the necessity of astrocytes in memory function (), we sought to test whether astrocytic activation can enhance memory performance. Mice were injected bilaterally with AAV8-GFAP::hM3Dq-mCherry to the dorsal CA1. To verify astrocytic activation in vivo, we administered CNO (3 mg/kg, intraperitoneally [i.p.]) 3 weeks after surgery, and brains were collected 90 min later and stained for the immediate-early gene cFos. CNO dramatically increased cFos levels in astrocytes of hM3Dq-expressing mice, compared to saline-injected controls ( Figures 4 A and 4B ; p < 0.0005, t test). Fear conditioning (FC) exposure had no effect on cFos levels in astrocytes ( Figure S4 A).

(F) No effect on auditory-cued memory was observed, with both groups showing increased freezing during tone presentation (p < 0.00001). Data presented as mean ± SEM.

(D and E) CNO application did not affect the exploration of the conditioning cage (D) but resulted in a > 40% improvement in contextual freezing in CNO-treated mice tested one day later, compared to saline treated controls (p < 0.005) (E).

(C) CNO application had no effect on exploration of the maze. Similarly, CNO had no effect on the exploration of the FC cage before conditioning. To replicate the results presented in Figures 4 E and 4F, mice expressing hM3Dq in their CA1 astrocytes were injected with either saline (n = 8) or CNO (n = 10) 30 minutes before fear conditioning acquisition.

(B) Mice expressing hM3Dq in their CA1 astrocytes were injected with either saline (n = 7) or CNO (n = 7) 30 minutes before T-maze training.

(J) In a novel environment, saline-treated (n = 6) and CNO-treated (n = 6) hM3Dq mice explored the field with similar path lengths (top), and there was no effect on anxiety (bottom), because the percentage of time that saline- and CNO-treated mice spent in the center of the open field was similar. Representative exploration traces are presented. Data are presented as mean ± SEM.

(H and I) In a new group of GFAP::hM3Dq mice, CNO administration (n = 7) only during the recall test had no effect on either contextually (H) or auditory-cued (I) memory, compared to saline-injected controls (n = 7) with both groups showing increased freezing during tone presentation ( ∗ p < 0.000001).

(G) Astrocytic activation by CNO application during retrieval on the next day did not further alter recall, but the original improvement was maintained ( ∗ p < 0.05).

(F) Astrocytic activation in CA1 had no effect on auditory-cued memory in a novel context, with both groups showing increased freezing during tone presentation ( ∗ p < 0.00001).

(E) GFAP::hM3Dq mice were injected with saline (n = 6) or CNO (n = 7) 30 min before fear conditioning acquisition. CNO application before training resulted in a 40% improvement in contextual freezing in mice tested 1 day later, compared to saline-treated controls ( ∗ p < 0.015).

(C) GFAP::hM3Dq mice that were injected with CNO (n = 7) 30 min before T-maze training demonstrated > 30% improved novel arm recognition ( ∗ p < 0.05) compared to their saline-injected controls (n = 7).

(A and B) (A) CNO administration in vivo to mice expressing hM3Dq (red) in CA1 astrocytes resulted in a significant increase in cFos expression (green) in the activated astrocytes, compared to saline-injected controls ( ∗ p < 0.0005; n = 4 mice, 18 slices, for both groups; scale bar, 50 μm). (B) Representative images to the group data presented in (A).

Release of gliotransmitters through astroglial connexin 43 hemichannels is necessary for fear memory consolidation in the basolateral amygdala.

Here, we show, for the first time, that astrocytic activation is sufficient to induce de novo potentiation of CA3 to CA1 synapses, and that this potentiation is long lasting, beyond the time of astrocytic activation. The fact that our manipulation produced prolonged potentiation whereas Cauncaging in astrocytes required additional direct post-synaptic depolarization to induce LTP (), may be due to the prolonged astrocytic activation in our experiments. Furthermore, we show that the synaptic potentiation induced by astrocytic activation is mediated by the NMDA receptor, similar to classical CA3 to CA1 LTP, and demonstrate the involvement of the NMDA co-agonist D-serine in this potentiation.

Finally, to confirm that synaptic potentiation induced by astrocytic activation is mediated by an increase in astrocytic intracellular Calevels, we applied CNO after filling a group of astrocytes with the Cachelator EGTA and CaCl, to clamp intracellular free Caat a steady-state concentration of 50–80 nM ( Figure S3 D–S3G). fEPSPs recorded in adjacent neurons were only minimally potentiated compared to the significant potentiation observed in distant neurons ( Figure S3 H).

We subsequently tested the role of metabotropic glutamate receptors (mGluRs) in astrocyte-induced synaptic potentiation, and found that the CNO application significantly potentiated fEPSPs even in the presence of the mGluRs blockers 2-methyl-6-(phenylethynyl)pyridine (MPEP, 50 μM) and LY367385 (100 μM) ( Figures 3 G–3I, orange), suggesting no contribution of mGluRs in the observed potentiation (pairwise comparisons: CNO to baseline p < 0.0005, washout to baseline p < 0.01) ( Figure 3 I).

D-serine was shown to underlie the necessity of astrocytes to LTP (). To resolve the role of D-serine in astrocyte-induced de novo potentiation, we used the NMDA D-serine site blocker 5,7-dichlorokynurenic acid (DCKA) (750 nM; cyan), which completely blocked potentiation ( Figures 3 G–3I). We then repeated the experiment in the presence of 10 μM D-serine in the bath and found that when the D-serine co-agonist site was highly occupied, the effect of CNO was occluded, and no potentiation was observed ( Figure S3 C).

To perturb the mechanism underlying this long-term plasticity, we repeated the experiment with the NMDA receptor blocker APV (50 μM; green) applied to the recording chamber, in which case CNO application failed to increase fEPSP size ( Figures 3 G–3I). Another demonstration of the involvement of NMDA in astrocyte-induced potentiation is the finding that in the presence of higher magnesium levels (2 mM, compared to 1 mM in the original experiment), more SC stimulations were necessary to reach the full potentiation effect (time effect, F= 5.85 p < 0.02, pairwise comparisons: baseline to CNO11-20 p < 0.05, baseline to CNO21-30 p < 0.005) ( Figure S3 B).

The necessity of astrocytes in neuronal plasticity was repeatedly demonstrated in brain slices () and in vivo (). Furthermore, Cauncaging in astrocytes combined with post-synaptic depolarization (but neither of these manipulations alone) has been reported to induce long-term potentiation (LTP) (), and repeated depolarization of DG astrocytes increased evoked EPSCs amplitude in granular neurons (). We examined the effect of astrocytic Gq activation on evoked synaptic events in CA1 neurons in response to Schaffer collaterals (SCs) stimulation before and after bath application of CNO ( Figures 3 A and 3B ). Surprisingly, we observed a 50% potentiation of the EPSC amplitude in response to the same stimulus in GFAP::hM3Dq slices treated with CNO (crimson), but not in slices from the same mice exposed to ACSF (blue) only (time-by-treatment interaction F= 3.158, p < 0.00005) ( Figures 3 C and 3D). To verify that this effect cannot be attributed to the application of CNO per se, we injected mice with a control AAV8-GFAP::mCherry virus ( Figure S2 E), and found no alteration in the response to stimulation before and after CNO application in slices from these mice ( Figure S3 A). To examine the long-term persistence of the observed potentiation, we tested the effect of astrocytic Gq activation on the evoked field potential in CA1 stratum radiatum to SC stimulation before, during, and after astrocytic activation ( Figures 3 E and 3F). We started by replicating our finding of de novo potentiation following astrocytic manipulation, now in the network level. This was shown by a greater than 150% increase in field EPSP (fEPSP) amplitude in response to a given stimulus in GFAP::hM3Dq slices treated with CNO, but not in ASCF-treated slices ( Figures 3 G–3I). We then washed the CNO out of the recording chamber for 20 min and measured fEPSPs for 10 more min. Evoked responses remained significantly potentiated in GFAP::hM3Dq slices that were previously treated with CNO, but not in ACSF-treated slices ( Figures 3 G–3I) (time-by-treatment interaction, F= 3.16, p < 0.0000005, Figure 3 H; time-by-treatment interaction F= 3.48, p < 0.005, pairwise comparisons: CNO to baseline p < 0.000005, washout to baseline p < 0.0005, Figure 3 I).

(D–H) blocking Ca 2+ increase in astrocytes prevents CNO-induced neuronal potentiation: In an area of hM3Dq-expressing (red) astrocytes (D), an astrocyte was patched with an internal solution containing the Ca 2+ chelator EGTA and Alexafluor488 (green), which then diffused into neighboring astrocytes (E). We verified that the patched cells are astrocytes based on morphology, and on their non-excitable responses to stimulation in both current-clamp and voltage clamp. (F and G) Specifically, we injected step current in steps of 100 pA from −500 pA to +500 pA (F), and step voltage in 5mV steps, from −90 mV to +90 mV (G), and recorded typical non-excitable responses. When astrocytic activity was blocked in a group of astrocytes adjacent to the field recording site (n = 3 slices from 3 mice) only a minimal potentiation compared to ACSF-treatment was observed. (H) When the field response was recorded at a distant site from the blocked astrocytes, a full potentiation to > 250% of baseline was observed. Representative traces are presented in corresponding colors. Data presented as mean ± SEM.

(C) Astrocytic activation by CNO failed to induce synaptic potentiation when 10 μM D-Serine was present in the recording bath (n = 7 slices from 3 mice).

(B) Astrocytic activation by CNO in the presence of 2mM magnesium in the recording chamber induced a > 130% potentiation in evoked fEPSP amplitude compared to slices from the same mice treated with ACSF, but required more SC stimulations or time to reach that effect, compared to the same manipulation performed with 1mM Magnesium (n = 3 slices from 3 mice; Time effect F(3,8.1) = 5.85, p < 0.02; Pairwise comparisons: Baseline versus 11-20 stimuli, p < 0.05; Baseline versus 21-30 stimuli, p < 0.005; 1-10 versus 21-30 stimuli, p < 0.05). Representative traces are presented in corresponding colors.

(A) CNO application per se has no effect on evoked EPSCs in response to Schaffer Collaterals (SC) stimulation in mCherry-expressing slices.

De Novo Potentiation Induced by Astrocytic Activation Is Occluded by D-Serine and Blocked by CaChelation in Astrocytes, Related to Figure 3

(H) Astrocytic activation by CNO induced >150% potentiation in evoked fEPSP amplitude compared to slices from the same mice treated with ACSF. Application of the NMDA receptor blocker APV and the D-serine co-agonist site blocker DCKA completely eliminated the astrocyte-induced potentiation, but the mGluR blockers MPEP and LY367385 had no effect (CNO, n = 4–5; CNO+APV, n = 3; CNO+DCKA, n = 6; CNO+MPEP+LY, n = 4; ACSF, n = 2).

(G) Overlaid evoked fEPSPs from ACSF (blue), CNO (crimson), CNO+APV (green), CNO+CDKA (cyan), and CNO+MPEP+LY367385 (orange) treated slices before and after drug application and after drug washout.

(F) Measurements were performed for 10 min at three time points: before CNO application, 15 min after CNO application, and 20 min after CNO washout.

(E) Extracellular field recordings from the apical dendrites of CA1 hippocampal neurons surrounded by hM3Dq-expressing astrocytes were performed in response to SC stimulation.

(D) No differences in evoked EPSCs amplitude were observed between the groups before drug application. Astrocytic activation by CNO induced a prolonged potentiation in evoked EPSC amplitude compared to slices from the same mice treated with ACSF (CNO, n = 8; ACSF, n = 4; p < 0.00005).

(C) Overlaid evoked EPSCs from an ACSF-treated cell (blue) and a CNO-treated cell (crimson) before and after drug application are presented (single events are in gray; averages are bolded).

(B) Measurements were performed for 10 min at two time points: before CNO application or 15 min after CNO application.

We provide the first demonstration that astrocytic population activation increases both the rate and the potency of spontaneous miniature events impinging on CA1 pyramidal cells. It is possible that by using stimulation that more closely mimics physiological astrocytic activity, compared to repeated electrical depolarizations () or mechanical stimulation (), we have been able to unveil the more subtle amplitude potentiation effect.

Calcium elevation in astrocytes causes an NMDA receptor-dependent increase in the frequency of miniature synaptic currents in cultured hippocampal neurons.

Manipulations of single astrocytes have been employed in the past to show their involvement in spontaneous release events () and in excitatory post-synaptic potential (EPSP) induction success rate of minimal stimulation in the hippocampus (). Specifically, astrocytic inhibition resulted in more EPSP failures (), whereas astrocytic activation increased both the frequency of miniature spontaneous events and the responses to minimal stimulation (), with no effect on amplitude, suggesting an exclusive pre-synaptic influence. To examine the effect of Gq pathway activation restricted to CA1 astrocytes on spontaneous synaptic release, we performed whole-cell recordings from CA1 hippocampal neurons in mice expressing hM3Dq in CA1 astrocytes ( Figure 2 A). Recordings were performed at a depth of ∼100 μm in the slice, where the full structure of both the recorded neuron and the surrounding astrocytes is preserved. We recorded spontaneous release events in voltage clamp under tetrodotoxin (TTX) (1 μM) before and after bath application of CNO (10 μM), and found that CNO application resulted in increased frequency of miniature excitatory post-synaptic currents (mEPSCs) ( Figures 2 B–2E, S2 A, and S2B; p < 0.00001, Kolmogorov-Smirnov test, and p < 0.00001, t test for the average change per cell). Importantly, astrocytic activation also induced a significant increase in mEPSC amplitude ( Figures 2 B, 2C, 2F, 2G, S2 C, and S2D; p < 0.0001, Kolmogorov-Smirnov test, and p < 0.01, t test for the average change per cell), compared to slices from the same mice treated with ACSF only. To verify that CNO application itself does not produce similar effects, we injected mice with a control virus (AAV8-GFAP::mCherry) ( Figure S2 E). In hippocampal slices from these mice, neither ACSF nor CNO had an effect on mEPSC frequency ( Figures S2 F and S2G) or amplitude ( Figures S2 H and S2I).

(F–I) Neither ACSF nor CNO in mCherry expressing slices had any effect on either the frequency (F and G) or the amplitude (H and I) of spontaneous miniature events.

(E) Bilateral double injection of AAV8-GFAP-mCherry resulted in mCherry expression in CA1 astrocytes. Scale bar 100 μm. Whole-cell voltage-clamp recordings from CA1 hippocampal neurons surrounded by mCherry expressing astrocytes were performed before and after ACSF or CNO bath application (4 slices, from 2 mice and 4 slices from 3 mice, respectively).

(D) A significant increase in mEPSC amplitude was observed after, compared to before, CNO application in hM3Dq slices (p < 0.001).

(C) No change in the amplitude of spontaneous miniature events was observed in hM3Dq mice before and after ACSF application.

(B) A significant increase in mEPSC frequency was observed after, compared to before, CNO application in hM3Dq slices (p < 0.001).

(A) No change in the frequency of spontaneous miniature events was observed in hM3Dq mice before and after ACSF application.

(G) CNO application increased the mEPSC amplitude compared to ACSF alone (p < 0.0001). Inset: average change in amplitude for all recorded cells ( ∗ p < 0.01). Data are presented as mean ± SEM.

(F) No differences in mEPSC amplitude were observed between the groups before drug application.

(E) CNO application increased the frequency of spontaneous events, compared to ACSF alone (p < 0.00001). Inset: average change in frequency for all recorded cells ( ∗ p < 0.00001).

(D) No differences in mEPSCs frequency were observed between the groups before drug application.

(C) Overlaid events from 5-min-long traces in two representative cells. Single events are in gray, and averages are bolded.

(B) Spontaneous miniature release events under TTX were recorded before and after CNO application (CNO, n = 6; ACSF, n = 3). Sample traces of an ACSF-only cell (blue) and a CNO-treated cell (crimson) are shown.

Calcium elevation in astrocytes causes an NMDA receptor-dependent increase in the frequency of miniature synaptic currents in cultured hippocampal neurons.

To verify that hM3Dq activates astrocytes upon CNO application, we performed two-photon calcium (Ca) imaging in brain slices. CA1 astrocytes expressing both hM3Dq and GCaMP6f were identified, and a glass pipette was placed adjacent to locally apply CNO (10 mM) ( Figure 1 H). CNO application triggered an intracellular Caincrease in hM3Dq-expressing astrocytes, whereas artificial cerebro spinal fluid (ACSF) application had no effect (p < 0.005, t test) ( Figures 1 I–1K). To characterize the effects of CNO application on astrocytic activity over longer time durations, relevant for upcoming slice and in vivo experiments, we imaged brain slices with CA1 astrocytes co-expressing GCaMP6f and mCherry ( Figures S1 A and S1B) before and during CNO application (10 μM), and then after CNO washout ( Figure S1 C). CNO application to the imaging chamber induced a significant increase in the number of Catransients in hM3Dq-GCaMP6f astrocytes ( Figures S1 D and S1E) that lasted 40 min. After CNO washout, the transient frequency returned to baseline. Importantly, ACSF application to hM3Dq-GCaMP6f slices or CNO application to GCaMP6f-only slices had no effect on Catransients (time-by-group effect F(4,74) = 2.73, p < 0.05, post hoc pairwise comparisons CNO versus ACSF and GCaMP alone during manipulation p < 0.005 and p < 0.001, respectively; CNO manipulation versus CNO baseline and CNO washout p < 0.005 and p < 0.0005, respectively). To conclude, hM3Dq is specifically expressed in CA1 astrocytes and can trigger an increase in intracellular Caand in the frequency of Caevents, two markers for astrocytic activity (), upon CNO application.

(D and E) CNO application to hM3Dq-expressing slices (crimson) significantly increased the number of astrocytic events, compared to hM3Dq slices treated with ACSF (blue) (p < 0.005), to CNO application to slices with no hM3Dq (green) (p < 0.001), and to CNO baseline (p < 0.005) and CNO washout (p < 0.0005). Activity after CNO washout returned to baseline levels.

(C) 2-photon imaging was performed for 5 minutes before drug application (Baseline), and then the drug was applied. 15 minutes later, activity was imaged 3 times for 5 minutes with a 5 minutes interval between imaging sessions. The drug was then washed out, and 20 minutes later two 5 minutes imaging session, with a 5 minutes interval were performed.

(A and B) CA1 astrocytes co-expressing hM3Dq-mCherry in their membranes and GCaMP6f in their cytoplasm (scale bar 30 μm) were imaged.

Based on the ability of endogenous Gq-GPCRs to induce Caelevation in astrocytes () and their importance in neuron-astrocyte communication (), we chose to express the Gq-coupled designer receptor hM3Dq () in CA1 astrocytes, allowing their time-restricted activation by clozapine-N-oxide (CNO). As the CA1 region of the hippocampus has been repeatedly shown to be involved in contextual memory (), and neurons in this region have demonstrated learning-dependent potentiation (), we delivered an adeno-associated virus serotype 8 (AAV8) vector encoding hM3Dq fused to mCherry under the control of the astrocytic glial fibrillary acidic protein (GFAP) promoter (AAV8-GFAP::hM3Dq-mCherry) to this region ( Figure 1 A). Within the virally transduced region, hM3Dq expression was limited to the astrocytic outer membranes ( Figure 1 B), with high penetrance (>97% of the GFAP cells expressed hM3Dq) ( Figure 1 C) and almost complete specificity (>97% hM3Dq positive cells were also GFAP positive) ( Figure 1 D). Co-staining with the microglial marker Iba1 showed no overlap with hM3Dq expression ( Figure 1 E). The staining for neuronal nuclei (NeuN) revealed no co-localization with hM3Dq ( Figure 1 F), but served to illustrate how astrocytic processes within CA1 enwrap their neighboring neurons ( Figure 1 G).

(K) Summary of the results from all imaged cells (CNO n = 5 cells, ACSF n = 2 cells, from 3 mice) showing a significant increase in CNO-treated cells only ( ∗ p < 0.005). Data are presented as mean ± SEM.

(J) Traces showing the change in fluorescence (in Δf/f) from hM3Dq-expressing astrocytes treated with CNO (crimson) or ACSF (blue). A gray line represents the 200 ms of CNO application. The top trace is from the astrocyte shown in (H) and (I).

(I) Local CNO administration via the glass pipette containing Alexa 594 (red) in the vicinity of the same astrocyte induced an increase in intracellular Ca 2+ .

(E and F) No co-localization with the microglia marker Iba1 (E) or the neuronal nuclear marker NeuN (F) was detected (scale bar, 50 μm).

(C and D) GFAP::hM3Dq was expressed in >97% of CA1 astrocytes (408/419 cells from 3 mice; C), with >97% specificity (408/419 cells from 3 mice; D).

(B) hM3Dq (red) was expressed in the astrocytic membrane around the soma and in the distal processes (scale bar, 50 μm).

Discussion

In recent years, it has become clear that astrocytes play an important role in neuronal activity and plasticity. In this work, we used advanced tools to specifically manipulate hippocampal astrocytes, and show for the first time that astrocytic activation can dramatically potentiate synaptic transmission, promote memory allocation, and improve memory performance. Specifically, even in the absence of any direct potentiating protocol delivered to the neurons, chemogenetic astrocytic activation generated de novo, sustainable, NMDA-dependent potentiation of CA3 to CA1 synapses. In vivo, astrocytic activation increased neuronal activity in a task-dependent manner only in learning mice, and enhanced cognitive performance in two memory tests. Finally, we show that optogenetic astrocytic activation precisely during memory acquisition induced a drastic memory enhancement.

Kheirbek et al., 2013 Kheirbek M.A.

Drew L.J.

Burghardt N.S.

Costantini D.O.

Tannenholz L.

Ahmari S.E.

Zeng H.

Fenton A.A.

Hen R. Differential control of learning and anxiety along the dorsoventral axis of the dentate gyrus. Yizhar et al., 2011 Yizhar O.

Fenno L.E.

Prigge M.

Schneider F.

Davidson T.J.

O’Shea D.J.

Sohal V.S.

Goshen I.

Finkelstein J.

Paz J.T.

et al. Neocortical excitation/inhibition balance in information processing and social dysfunction. Han et al., 2007 Han J.H.

Kushner S.A.

Yiu A.P.

Cole C.J.

Matynia A.

Brown R.A.

Neve R.L.

Guzowski J.F.

Silva A.J.

Josselyn S.A. Neuronal competition and selection during memory formation. Yiu et al., 2014 Yiu A.P.

Mercaldo V.

Yan C.

Richards B.

Rashid A.J.

Hsiang H.L.

Pressey J.

Mahadevan V.

Tran M.M.

Kushner S.A.

et al. Neurons are recruited to a memory trace based on relative neuronal excitability immediately before training. Josselyn et al., 2015 Josselyn S.A.

Köhler S.

Frankland P.W. Finding the engram. Conversely, we show that directly increasing the activity of CA1 neurons dramatically impairs memory acquisition. This finding is in agreement with optogenetic studies showing that unselective activation of dorsal DG or prefrontal cortex neurons during FC acquisition compromises recall on the next day (). However, pre-training neuronal activation is not necessarily detrimental. For example, when the activity of a small, selective neuronal population in the BLA is increased before FC acquisition, a beneficial effect is conferred (). Neurons active before training are also more likely to be allocated to the engram supporting the acquired memory ().

Why then, does broad non-selective astrocytic activation enhance memory, whereas similar neuronal activation impairs it? Our results show a tailored response of astrocytes to the activity of their surrounding neurons, resulting in task-specific increase in neuronal activity, only in the ensemble active during memory allocation. Contrary to the non-selective increase in activity following chemogenetic stimulation of neurons, astrocytic activation does not increase baseline neuronal activity in the absence of an additional salient stimulus.

Allen et al., 2017 Allen W.E.

DeNardo L.A.

Chen M.Z.

Liu C.D.

Loh K.M.

Fenno L.E.

Ramakrishnan C.

Deisseroth K.

Luo L. Thirst-associated preoptic neurons encode an aversive motivational drive. Liu et al., 2012 Liu X.

Ramirez S.

Pang P.T.

Puryear C.B.

Govindarajan A.

Deisseroth K.

Tonegawa S. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Ye et al., 2016 Ye L.

Allen W.E.

Thompson K.R.

Tian Q.

Hsueh B.

Ramakrishnan C.

Wang A.C.

Jennings J.H.

Adhikari A.

Halpern C.H.

et al. Wiring and molecular features of prefrontal ensembles representing distinct experiences. This selective effect of astrocytes brings to mind state of the art genetic tools that now offer activity-dependent neuronal targeting, allowing the specific tagging and manipulation of ensembles that were active during a specific time window (). Like these tools, astrocytes, monitoring both the input and output information in their surrounding neuronal network, can detect and specifically enhance neuronal activity in response to a meaningful stimulus (such as SC stimulation in slice or FC in vivo), thus providing a similar activity-dependent specificity. This could explain why astrocytic activation improves memory performance, whereas neuronal activation impairs it.

Josselyn et al., 2015 Josselyn S.A.

Köhler S.

Frankland P.W. Finding the engram. Rogerson et al., 2014 Rogerson T.

Cai D.J.

Frank A.

Sano Y.

Shobe J.

Lopez-Aranda M.F.

Silva A.J. Synaptic tagging during memory allocation. Lee and Silva, 2009 Lee Y.S.

Silva A.J. The molecular and cellular biology of enhanced cognition. To summarize, memory performance is not a simple binary process (remember/not remember), but can vary greatly both between and within memories. Memory ensemble allocation and maintenance depend on the activity level of neurons (), which may well be contingent on their environment. As biological processes do not always reach their potential maxima, it is tempting to devise ways to enhance normal memory performance (). Here, we show that activating astrocytes in mice with intact cognition improves their memory acquisition. In light of the justified hesitance to directly increase general neuronal activity, our finding that astrocytic modulation can enhance memory acquisition without affecting basal neuronal activity may have important clinical implications for cognitive augmentation treatments. The major advantage of using astrocytic modulation for this purpose is that the specificity of the effect is conferred by the astrocytes, not by the method of external manipulation, allowing straightforward translation to pharmacology.

Importantly, the capacity of astrocytes to independently induce plasticity and improve cognitive performance, as reported here, suggests that astrocytes can autonomously compute task-specific information based on the surrounding neuronal activity, which they then communicate back to the neuronal circuit. This perspective calls for a reassessment of the view of neuro-glia interaction, expanding the limited role of astrocytes as support cells merely enabling plastic and cognitive processes, to a broader function of these cells in actively shaping neuronal networks.