Astrocytes gate long-term potentiation in hippocampal interneurons
Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Hangzhou City University, Hangzhou, 310015, China
Institute of Pharmacology & Toxicology, College of Pharmaceutical Sciences, Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Zhejiang University, Hangzhou, 310058, China
Zhejiang Key Laboratory of Neuroelectronics and Brain Computer Interface Technology, Hangzhou, 311121, China
School of Medicine, Nankai University, Tianjin, 300071, China
Department of Neurology, The Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, 310052, China
*Corresponding authors: Ling-Hui Zeng (zenglh@hzcu.edu.cn), Weida Shen (shenwd@hzcu.edu.cn)Abstract
Long-term potentiation is involved in physiological processes such as learning and memory, motor learning and sensory processing, and pathological conditions such as addiction. In contrast to the extensive studies on the mechanism of long-term potentiation on excitatory glutamatergic synapses onto excitatory neurons (LTPE→E), the mechanism of LTP on excitatory glutamatergic synapses onto inhibitory neurons (LTPE→I) remains largely unknown. In the central nervous system, astrocytes play an important role in regulating synaptic activity and participate in the process of LTPE→E, but their functions in LTPE→I remain incompletely defined., We studied the role of astrocytes in regulating LTPE→I in the hippocampal CA1 region and their impact on cognitive function using electrophysiological, pharmacological, confocal calcium imaging, chemogenetics and behavior tests. We showed that LTPE→I in the stratum oriens of hippocampal CA1 is astrocyte independent. However, in the stratum radiatum, synaptically released endocannabinoids increase astrocyte Ca2+ via type-1 cannabinoid receptors, stimulate D-serine release, and potentiate excitatory synaptic transmission on inhibitory neurons through the activation of (N-methyl-D-aspartate) NMDA receptors. We also revealed that chemogenetic activation of astrocytes is sufficient for inducing NMDA-dependent de novo LTPE→I in the stratum radiatum of the hippocampus. Furthermore, we found that disrupting LTPE→I by knocking down γCaMKII in interneurons of the stratum radiatum resulted in dramatic memory impairment. Our findings suggest that astrocytes release D-serine, which activates NMDA receptors to regulate LTPE→I, and that cognitive function is intricately linked with the proper functioning of this LTPE→I pathway.
Article notes
Competing Interest Statement
The authors declare no competing interests.
Summary of Updates:
Introduction
Long-term potentiation (LTP) was originally identified as a long-term increase in synaptic connections at glutamatergic synapses onto granule cells of the hippocampus (Bliss & Lomo 1973). This form of plasticity has been well documented in CA1 pyramidal cells, where synaptic plasticity can be induced by the activation of postsynaptic (N-methyl-D-aspartate) NMDA receptors (NMDARs), voltage-dependent Ca2+ channels, and metabotropic glutamate receptors (mGluRs) (Bliss & Collingridge 1993; Nicoll 2017). Numerous studies have provided compelling evidence that LTP in the hippocampal network occurs not only at excitatory glutamatergic synapses onto excitatory pyramidal and granule cells (LTPE→E) but also at excitatory glutamatergic synapses onto inhibitory interneurons (LTPE→I) (Kullmann & Lamsa 2007; Kullmann & Lamsa 2011; Asgarihafshejani et al. 2022; Le Duigou et al. 2015; Pelletier & Lacaille 2008). However, the mechanism underlying LTPE→I remains controversial due to the heterogeneous types of interneurons in the hippocampus and the absence of synaptic spines between excitatory inputs on interneurons (Pelkey et al. 2017; Kullmann & Lamsa 2007). Two distinct forms of LTPE→I, namely NMDAR-dependent LTPE→I and NMDAR-independent LTPE→I, have been observed in hippocampal interneurons (Kullmann & Lamsa 2007; Kullmann & Lamsa 2011).
Astrocytes are the most prevalent glial cell type in the central nervous system (CNS) and play a critical role in regulating the development and function of the nervous system (Escartin et al. 2019; Verkhratsky & Nedergaard 2018; Chaboub & Deneen 2013; Perez-Catalan et al. 2021). Astrocytes have multipolar branches with numerous microprocesses that allow them to closely associate with blood vessels, neuronal cell bodies and axons, other glial cells and synapses (Bushong et al. 2002; Xie et al. 2022; Santello et al. 2019). Astrocytes also express various ion channels, transporters and neurotransmitter receptors (Ciappelloni et al. 2017; Verkhratsky & Steinhäuser 2000; Verkhratsky & Nedergaard 2018). With these membrane proteins, astrocytes can sense neuronal activity and exhibit increases in intracellular Ca2+ in reaction to neurotransmitters, and in turn, they release neuroactive chemicals called gliotransmitters that regulate synaptic transmission and plasticity (Araque et al. 2014; Bazargani & Attwell 2016; Khakh & McCarthy 2015; Verkhratsky & Nedergaard 2018), but also please see refer to Hamilton and Attwell 2010 (Hamilton & Attwell 2010). Numerous studies have shown that the release of D-serine, a co-agonist of NMDAR, from astrocytes is capable of enabling LTP in cultures, in slices and in vivo (Henneberger et al. 2010; Robin et al. 2018; Yang et al. 2003; Mothet et al. 2006; Panatier et al. 2006). Given the growing number of studies demonstrating the direct roles that astrocytes play in regulating LTPE→E, understanding whether and how astrocytes modulate LTP of excitatory postsynaptic currents in interneurons is of particular interest.
In this particular study, our focus was on the interneurons distributed in the stratum radiatum layer of the CA1 region of the hippocampus. Approximately 80% of these interneurons exhibit NMDAR-dependent LTPE→I when presynaptic stimulation is paired with postsynaptic depolarization. We found that blocking astrocyte metabolism and clamping of astrocyte Ca2+ signaling can prevent LTPE→I in large NMDAR-containing interneurons, which could be rescued by bath application of D-serine. Furthermore, pharmacological and Ca2+ imaging studies have shown that astrocytes respond to Schaffer collateral stimulation with Ca2+ increases through activation of type-1 cannabinoid receptors (CB1Rs), which stimulate the release of D-serine and further regulate LTPE→I via binding to the glycine site of NMDARs. We also found that activating astrocytes with Gq designer receptors exclusively activated by designer drugs (DREADDs) induced a potentiation of excitatory to inhibitory synapses in the stratum radiatum. Additionally, knockdown of γCaMKII hampered LTPE→I in the stratum radiatum of the hippocampus in brain slices and disrupted contextual fear conditioning memory in vivo. Taken together, these results are the first to indicate that astrocytes are an integral component of a form of long-term synaptic plasticity between glutamatergic neurons and GABAergic interneurons, and that memory is also regulated by LTPE→I.
Results
Astrocytes play a role in the formation of NMDAR-dependent LTPE→I in the CA1 stratum radiatum
To visualize CA1 stratum radiatum interneurons, we delivered an adeno-associated virus serotype 2/9 (AAV2/9) vector encoding EGFP under the control of the interneuronal mDLx promoter (AAV2/9-mDLx-EGFP) to this region (Dimidschstein et al. 2016). Within the virally transduced region, EGFP expression was limited to the interneuron, with high penetrance (>98% of the GAD67 cells expressed EGFP) (Figure 1-figure supplement 1A-C) and almost complete specificity (>98% EGFP-positive cells were also GAD67 positive) (Figure 1-figure supplement 1D). These immunostaining results indicate that EGFP expression was limited to the interneurons in the CA1 region of the stratum radiatum.
We next examined whether expressing EGFP in interneurons affects their membrane properties and synaptic transmission. Therefore, we performed whole-cell patch recordings of EGFP+ interneurons and putative interneurons in the CA1 stratum radiatum of the hippocampus in the control mice in the presence of the GABAA receptor blocker picrotoxin. We found that the excitability and resting membrane potential were not different between these EGFP+ interneurons and putative interneurons (Figure 1-figure supplement 2A-C). Moreover, we also found no difference in spontaneous excitatory postsynaptic currents (sEPSCs) or the paired-pulse ratio (PPR) at 50 ms interpulse intervals between EGFP+ interneurons and putative interneurons (Figure 1-figure supplement 2D-H). These results indicate that AAV injection and exogenous protein expression in interneurons have no effect on the membrane properties and baseline synaptic transmission of interneurons.
To avoid some necessary ingredient for LTPE→I induction being diluted from the cytoplasm, a perforate patch-clamp was used to record EPSPs from CA1 stratum radiatum interneurons. After a 10 min baseline recording, we delivered theta burst stimulation (TBS) consisting of 100 Hz stimulation with 25 pulses delivered in six trains separated by 20-second intervals. TBS was applied to induce LTPE→I of the excitatory inputs to CA1 interneurons. The interneurons were depolarized to -10 mV using a voltage-clamp model during TBS delivery. Under this condition, we found that in 8 out of 10 cells LTPE→I was induced for at least 45 min (Figure 1A-C). We repatched 6 of these successful LTPE cells and randomly patched 2 EGFP+ cells in the stratum radiatum in the whole-cell voltage-clamp model. The results indicated that all cells showed a linear current-voltage (I-V) curve for AMPAR, as well as a significant component of NMDAR-mediated currents (Figure 1-figure supplement 3). In addition, we found that the induction of LTPE→I was completely blocked by the NMDAR blocker D-AP5 (Figure 1A-C). This result confirmed that LTPE→I in CA1 stratum radiatum interneurons is NMDAR dependent (Lamsa et al. 2005; Lamsa et al. 2007).
To investigate whether astrocytes were involved in NMDAR-dependent LTPE→I in CA1 stratum radiatum interneurons, we treated slices with fluoroacetate (FAC, 5 mM) to specifically block astrocyte metabolism (Swanson & Graham 1994; Henneberger et al. 2010). The results showed that the induction of LTPE→I was blocked by FAC but not in the control slice (Figure 1D-F). Next, we tested whether glial metabolism is involved in LTPE→I in CA1 stratum orient, which has been shown to depend on calcium-permeable AMPARs (CP-AMPARs) and metabotropic glutamate receptors (mGluRs) (Le Duigou et al. 2015). We found that the induction of LTPE→I is not blocked by FAC in the CA1 stratum oriens (Figure 1-figure supplement 4A-B). We repatched 6 of these cells (4 cells from the control group and 2 cells from the FAC-treated group) in whole-cell voltage-clamp mode and observed that they exhibited high rectification AMPARs and a small component of NMDAR-mediated current (Figure 1-figure supplement 5). This observation confirms findings from a previous study (Lamsa et al. 2007; Oren et al. 2009). Moreover, we found that the induction of LTPE→I is not blocked by D-AP5 in the CA1 stratum oriens (Figure 1-figure supplement 4). Overall, the results indicate that the formation of LTPE→I in the CA1 stratum radiatum is tightly regulated by astrocyte function. The results in the stratum oriens also exclude the possibility that FAC directly affects the metabolism of interneurons which inhibit the formation of LTPE→I.
It is commonly accepted that astrocytic calcium signaling plays a pivotal role in triggering the release of gliotransmitters and modulating synaptic transmission (Bazargani & Attwell 2016; De Pitta et al. 2016; Sancho et al. 2021; Navarrete et al. 2012; Goenaga et al. 2023). In addition, it is well documented that astrocytic calcium signaling is necessary for the formation of LTPE→E in the CA1 stratum radiatum (Henneberger et al. 2010; Robin et al. 2018). Thus, we tested whether intracellular Ca2+ signals are needed for the induction of LTPE in the CA1 stratum radiatum. We found that clamping of the astrocyte Ca2+ concentration significantly suppressed LTPE→I (Figure 2A-D). Consistent with the previous study on LTPE→E, the supply of D-serine (50 μM) fully rescued NMDAR-dependent LTPE→I (Figure 2D). For the control, when the intracellular Ca2+ concentration of astrocytes was not clamped but the astrocytes were recorded with a glass pipette, LTPE→I was indistinguishable from that induced without patching an astrocyte (Figure 2D). Overall, these results indicate that functional preservation of astrocytic metabolism and Ca2+ mobilization is critical for maintaining the induction of LTPE→I.
Astrocytic Ca2+ transients, induced by activation of astroglial cannabinoid type 1 receptors (CB1Rs), are involved in the regulation of LTPE→I formation
Accumulating evidence indicates that neuronal depolarization in the hippocampus induces astrocytic Ca2+ transients, which are mediated by the activation of astroglial CB1Rs (Eraso-Pichot et al. 2023; Noriega-Prieto et al. 2023; Navarrete & Araque 2008; Navarrete & Araque 2010; Navarrete et al. 2014). Moreover, astroglial CB1R-mediated Ca2+ elevation is necessary for LTPE→E in the CA1 region of the hippocampus (Robin et al. 2018). Therefore, we asked whether astroglial CB1R-mediated Ca2+ elevations are needed for hippocampal LTPE→I. We first analyzed whether TBS could evoke astrocytic Ca2+ transients via CB1Rs in the stratum radiatum of the hippocampus. In this study, GCaMP6f was used to analyze astrocytic Ca2+ signals, which were specifically expressed in astrocytes by using adeno-associated viruses of the 2/5 serotype (AAV 2/5) with the astrocyte-specific gfaABC1D promoter (Figure 3-figure supplement 1A). Furthermore, the expression was confirmed by immunohistochemistry. Within the virally transduced region, GCaMP6f-positive cells in the CA1 stratum radiatum were also positive for the astrocyte-specific marker GFAP (Figure 3-figure supplement 1A and C-D). Costaining with the neuron marker NeuN showed no overlap with GCaMP6f expression (Figure 3-figure supplement 1B, E). Consistent with previous studies (Sherwood et al. 2017; Robin et al. 2018), we found that TBS significantly increased Ca2+ signaling in astrocytes of hippocampal slices in the presence of picrotoxin and CGP55845 (Figure 3A-B and F). As predicted, the increase in Ca2+ signals after TBS was inhibited by the CB1 receptor inhibitor AM251 (2 μM) (Figure 3C-D and F). Previous studies have demonstrated that activation of [1-adrenoceptors increases calcium signals (Shen et al. 2021; Ding et al. 2013; Gordon et al. 2005; Bekar et al. 2008; Paukert et al. 2014; Oe et al. 2020) and triggers the release of D-serine release in the neocortex (Pankratov & Lalo 2015). However, our results show that [1-adrenoceptor receptors are not involved in the Ca2+ signal increase observed after TBS (Figure 3F).
Next, we explored whether CB1R-mediated Ca2+ elevation was accompanied by the formation of LTPE→I. We found that LTPE→I was significantly reduced in AM251-treated slices relative to that in controls (Figure 3G-I). Consistent with the above results shown in Figure 2D, LTPE→I was rescued by the addition of D-serine in AM251-treated slices
D-serine release from astrocytes potentiates the NMDAR-mediated synaptic response
Next, we explored the underlying mechanisms by which astrocytes control the formation of LTPE→I in the CA1 stratum radiatum. Our above results indicate that D-serine is a downstream signaling pathway of astrocyte Ca2+ signaling. D-serine, a co-agonist of NMDAR, can be released by astrocytes through Ca2+-dependent exocytosis and regulate the function of NMDAR. It has been shown that the occupancy of synaptic NMDAR co-agonist sites by D-serine is not saturated in CA1 pyramidal cells (Robin et al. 2018; Papouin et al. 2012). It has been shown that the NMDAR co-agonist site in CA1 pyramidal neurons is fully saturated during the dark phase, but this saturation dissipates to subsaturating levels during the light phase (Papouin et al. 2017a). However, the level of occupancy of synaptic NMDAR co-agonist sites by D-serine in interneurons of the CA1 stratum radiatum remains unclear. Furthermore, previous studies have demonstrated that NMDAR-dependent synaptic responses of interneurons in the stratum radiatum display a strong rundown effect under whole-cell mode (Lamsa et al. 2005). Thus, we recorded the NMDAR-mediated EPSPs from stratum radiatum interneurons in the perforated-patch configuration in Mg2+-free ACSF, which showed no rundown effect in this configuration (Figure 4A-B). Bath application of 50 μM D-serine enhanced the NMDAR-mediated EPSPs (Figure 4A-C), indicating that the level of D-serine in the excitatory to inhibitory synapse cleft was not saturated to occupy the co-agonist site. Subsequently, we employed a similar protocol in another study to test whether TBS could increase the extracellular level of D-serine and promote NMDAR-mediated synaptic responses (Henneberger et al. 2010). Our results indicated that NMDAR-mediated responses were transiently enhanced after one TBS, and the percentage of potentiation was reduced by pretreatment of hippocampal slices with 50 μM D-serine (Figure 4D-F). However, disrupting glial metabolism with FAC rendered the percentage of TBS-induced potentiation not significantly different for that in the group pretreated with 50 μM D-serine (Figure 4F). Furthermore, the percentage of potentiation in AMPAR-mediated responses after TBS was insensitive to bath application of D-serine, indicating that the enhancement of the NMDAR-mediated response by TBS was not due tochanges in release probability and cell excitability (Figure 4F). Overall, our results indicate that D-serine release from astrocytes potentiate NMDAR-mediated responses by binding the co-agonist site and regulating the formation of LTPE→I.
Chemogenetic activation of astrocytes induced E→I synaptic potentiation
The crucial role of astrocytes in LTPE→E has been extensively demonstrated in brain slices (Henneberger et al., 2010; Min and Nevian, 2012; Pascual et al., 2005; Perea and Araque, 2007; Suzuki et al., 2011) and in vivo (Robin et al. 2018). Previous studies have demonstrated that the activation of astrocytes through chemogenetics leads to potentiation at CA1 synapses in the hippocampus in the absence of high-frequency stimulation (Nam et al. 2019; Adamsky et al. 2018; Van Den Herrewegen et al. 2021). In this study, we aimed to investigate whether the activation of the astrocytic G protein-coupled receptor (GPCR) pathway could trigger the potentiation of E→I synaptic transmission. We utilized an adeno-associated virus serotype 5 (AAV2/5) vector encoding hM3Dq fused to mCherry for specific activation of astrocytes via clozapine-N-oxide (CNO, 5 μM). To ensure specific expression in astrocytes, the vector was also under the control of the astrocyte-specific gfaABC1D promoter (Figure 5-figure supplement 1).
To verify whether CNO application could evoke Ca2+ transients in astrocytes, we delivered AVV of hM3Dq along with AAV of GCaMP6f and conducted confocal Ca2+ imaging in brain slices. Our results showed that CNO application indeed induced an increase in intracellular Ca2+ levels in cells coexpressing hM3Dq and GCaMP6f (Figure 5A-D). These results suggest that the expression of hM3Dq is selective to astrocytes and can elicit a rise in intracellular Ca2+ levels upon administration of CNO. Subsequently, we investigated the impact of astrocytic Gq activation on evoked synaptic events in interneurons of the CA1 stratum radiatum that were induced by Schaffer collaterals stimulation, both before and after the administration of CNO. Interestingly, we observed that the EPSC amplitude was potentiated by 60% in response to the exact same stimulus in gfaABC1D::hM3Dq slices treated with CNO (Figure 5E-G), while no such potentiation was detected in slices obtained from the mice that were injected with a control virus (AAV2/5-gfaABC1D::mCherry) (Figure 5E-G).
Previous studies have indicated that the synaptic potentiation triggered by chemogenetic activation of astrocytes is mediated through the release of D-serine by astrocytes, resulting in the activation of NMDARs (Adamsky et al. 2018). To verify whether the astrocytic-induced synaptic potentiation between excitatory and inhibitory neurons is mediated by D-serine release from astrocytes and the subsequent activation of NMDARs, we conducted an experiment in which we administered CNO after blocking the NMDARs with D-AP5 or saturating glycine site of NMDARs with 50 μM D-serine. Our results showed that both the NMDAR blocker D-AP5 and 50 μM D-serine completely inhibited the potentiation in EPSP amplitude observed in response to CNO-induced astrocytic activation (Figure 5H-J). Our findings demonstrate, for the first time, that astrocytic activation alone can trigger de novo potentiation of synapses between excitatory and inhibitory neurons and that this potentiation is indeed mediated by the release of D-serine from astrocytes and subsequent activation of NMDARs.
LTPE→I in CA1 of the stratum radiatum is necessary for long-term memory formation
We have previously shown that GABAergic interneurons in the hippocampus express high levels of γCaMKII, while αCaMK[, βCaMK [and δCaMK [are expressed at a lower frequency (He et al. 2021). Additionally, studies have demonstrated that γCaMKII expressed in hippocampal parvalbumin-positive (PV+) interneurons and cultured hippocampal inhibitory interneurons is essential for the induction of LTPE→I (He et al. 2021; He et al. 2022). Specifically, in hippocampal PV+ interneurons, this protein is also vital for the formation of hippocampus-dependent long-term memory in vivo (He et al. 2021). Therefore, we asked whether astrocyte-gated LTPE→I in the CA1 stratum radiatum is involved in the hippocampus-dependent long-term memory formation. Several independent groups have provided compelling evidence suggesting that astroglial CB1R-mediated signaling pathways regulate excitatory synapses formed by excitatory neurons rather than excitatory synapses between excitatory neurons and interneurons (Fernandez-Moncada & Marsicano 2023; Eraso-Pichot et al. 2023; Noriega-Prieto et al. 2023; Kano et al. 2009; Navarrete & Araque 2008; Navarrete & Araque 2010; Han et al. 2012). Thus, manipulating this pathway may also affect excitatory synapses formed by excitatory neurons. To avoid this side effect, we specifically knocked downγCaMKII expression in inhibitory neurons of the CA1 stratum radiatum by delivering an adeno-associated virus serotype 2/9 (AAV2/9) vector encoding shRNAs against γCaMKII and EGFP under the control of an inhibitory neuron-specific promoter (AAV2/9-mDLx-EGFP-γCaMKII shRNA) through bilateral stereotactic injection. We observed that interneurons identified by the specific marker GAD67 were also positive for EYFP and therefore likely expressed shRNAs, which led to knockdown γCaMKII in these cells (Figure 6-figure supplement 1). To confirm that knockdown of γCaMKII could hamper the induction of LTPE→I, EYFP-positive interneurons in the CA1 stratum radiatum were recorded in perforated-patch mode. We found that knocking down γCaMKII in CA1 stratum radiatum interneurons impaired LTPE→I, but LTPE→I in the putative interneurons in the stratum radiatum of control mice was unimpaired (Figure 6A-C). In addition, robust LTPE→I was induced by TBS in EYFP-positive interneurons injected with AAV-mDLx-scramble shRNA into the CA1 stratum radiatum of the hippocampus (Figure 6C). Moreover, it is worth noting that the resting membrane potential, frequency and amplitude of sEPSCs, excitability and PPR recorded from γCaMKII knockdown interneurons did not differ from those recorded from putative interneurons and EYFP-positive interneurons (infected with scramble shRNA) (Figure 6-figure supplement 2). Taken together, these results indicate that knocking down γCaMKII from interneurons has no effect on synaptic transmission at baseline but impairs the induction of LTPE→I in these interneurons.
Next, we examined the behavioral consequences of destroying astrocyte-gated LTPE→I in the CA1 stratum radiatum. We analyzed contextual fear conditioning memory, which is associated with activation of the hippocampus. We found that no effect of γCaMKII knockdown on exploration of the context before conditioning was observed (Figure 6D-E). 24 hours after training, the mice were returned to the training box, and freezing was measured during the first 2 min. We found that γCaMKII knockdown mice showed a significant reduction in freezing during contextual conditioning (Figure 6F). Consistent with our previous study (He et al. 2021), γCaMKII knockdown in interneurons in the CA1 stratum radiatum did not produce a significant effect on freezing during tone conditioning at 28 h after training (Figure 6G). Taken together, our results strongly suggest that the astroglial CB1R signaling pathway gated LTPE→I in the CA1 stratum radiatum plays a vital role in hippocampus-dependent long-term memory.
Discussion
In the present study, we found that LTPE→I in the CA1 stratum radium is tightly controlled by D-serine release from astrocytes via the astroglial CB1R-mediated Ca2+ elevation. In addition, knockdown of γCaMKII by specific shRNA in interneurons in the CA1 stratum radium causes cognitive function deficits. Taken together, our data indicate that astrocyte-gated LTPE→I in the CA1 stratum radium plays a critical role in preserving normal cognitive function.
CB1Rs are widely expressed in various brain regions, including the hippocampus, and are detectable in presynaptic terminals (Castillo et al. 2012; Kano et al. 2009), postsynaptic terminals (Marinelli et al. 2009; Bacci et al. 2004), intracellular organelles (Jimenez-Blasco et al. 2020; Gutierrez-Rodriguez et al. 2018) and astrocytes (Ramon-Duaso et al. 2023; Noriega-Prieto et al. 2023; Eraso-Pichot et al. 2023; Fernandez-Moncada & Marsicano 2023; Robin et al. 2018; Han et al. 2012; Navarrete & Araque 2010; Navarrete & Araque 2008). However, the effects of astroglial CB1R-mediated signaling on synaptic transmission and plasticity are highly debated. It has been shown that exogenous administration of Δ9-tetrahydro-cannabinol (THC) leads to temporally prolonged and spatially widespread activation of astroglial CB1 receptors and triggers glutamate release, which activates postsynaptic NMDARs and induces LTD in the CA3–CA1 hippocampal synapses, resulting in working memory deficits (Han et al. 2012). Araque and colleagues reported that eCB released from depolarized CA1 pyramidal cells activates astroglial CB1Rs with in a shorter and localized manner and induces glutamate release, which activates lateral presynaptic mGluRs and induces LTP (Navarrete & Araque 2010; Navarrete & Araque 2008). In another study, Robin et al. found that high-frequency stimulation (HFS) of Schaffer Collateral induces LTP, which is gated by the activation of astroglial CB1Rs and the release of D-serine from astrocytes (Robin et al. 2018). These diverse consequences of CB1 receptor activation may be due to different neuronal activity patterns that induce eCB release and the different nature of the agonists. Interestingly, it has been discovered that individual hippocampal astrocytes are capable of releasing both ATP/adenosine and glutamate and that this release occurs in a time-dependent and activity-sensitive manner in response to neuronal interneuron activity (Covelo & Araque 2018). These findings suggest that the specific type and intensity of astrocyte stimulation plays a critical role in determining the downstream signaling pathways that are triggered by CB1R activation in astrocytes. Consistent with a previous study, we found that the activation of astroglial CB1Rs induces Ca2+ elevation and triggers the release of D-serine which binds to postsynaptic NMDARs and induces LTP formation (Robin et al. 2018). Our results provide evidence that astroglial CB1R-mediated signaling not only modulates the E→E synapses, but also regulates E→I synapses.
The precise mechanisms by which neurons and astrocytes differentially regulate D-serine levels are yet to be fully elucidated (Papouin et al. 2017b; Wolosker et al. 2017; Wolosker et al. 2016). However, astrocytes play a significant role in regulating the availability of D-serine. The enzyme serine racemase catalyzes the conversion of L-serine into D-serine, which was initially found in astrocytes and microglia in the mammalian brain (Panatier et al. 2006; Stevens et al. 2003; Wolosker et al. 1999). It should be highlighted that serine racemase has also been detected in neurons (Benneyworth et al. 2012; Miya et al. 2008; Dun et al. 2008). A study showed that, despite a significant reduction in SR protein levels in the brains of neuronal SR knockout mouse brains, the reduction in D-serine levels was minimal, suggesting that neurons are not the exclusive source of D-serine (Benneyworth et al. 2012) and that neurons may produce and release D-serine under certain conditions. Notably, activation of G protein-coupled receptors in astrocytes through chemogenetic methods leads to LTP, which relies on the release of D-serine from astrocytes and the activation of NMDARs (Van Den Herrewegen et al. 2021; Adamsky et al. 2018). Specifically, the release of D-serine, which is regulated by CB1R in astrocytes, is needed for Ca2+-dependent modulation of LTP in vivo (Robin et al. 2018), as well as the threshold and amplitude of dendritic spikes (Bohmbach et al. 2022). Moreover, recent studies have shown that conditional connexin double knockout (Hosli et al. 2022) or knockdown of α4nAChR (Ma et al. 2022) in astrocytes can decrease the extracellular concentration of D-serine, which in turn reduces NMDAR-dependent synaptic potentiation. These findings suggest that astrocytes are the primary source of D-serine, which plays a crucial role in modulating the function of NMDARs.
It is well established that LTPE→E observed in the CA1 region of the hippocampus is triggered by activation of NMDARs. However, LTPE→I is less studied than LTPE→E, but recent evidence suggests that it also involves the activation of NMDARs (He et al. 2021; Lamsa et al. 2007; Kullmann & Lamsa 2007; Kullmann & Lamsa 2011; Lamsa et al. 2005; Nissen et al. 2010). There have been reports of NMDAR-dependent LTPE→I in various regions of the brain, including the hippocampus and cortex (Kullmann & Lamsa 2007; Kullmann & Lamsa 2011). Our results suggest that different synaptic mechanisms are involved in the induction of LTPE→I in different subregions of the hippocampus. The stratum radiatum, where interneurons contain NMDARs, is known to be sensitive to NMDAR-dependent LTPE→I. In contrast, the stratum oriens, where interneurons contain CP-AMPARs, appears to rely on the activation of CP-AMPA receptors for LTPE→I induction. Notably, our findings suggest that astrocytes contribute to NMDAR signaling in the induction of LTPE→I in the stratum radiatum through the release of the co-agonist D-serine. Notably, our study found that prolonged activation of astrocytes via the Gq-DREADD pathway resulted in a substantial and persistent increase in Ca2+ events and significantly potentiated EPSP responses. This is consistent with earlier observations made by other groups regarding LTPE→E (Adamsky et al. 2018; Van Den Herrewegen et al. 2021). Above all, the mechanism of LTPE→I in the stratum radiatum appears to be shared by LTPE→E observed in the CA1 region.
A previous study demonstrated that knocking down γCaMKII from interneurons can disrupt LTPE→I and cognitive function (He et al. 2021; He et al. 2022). Our results confirmed that knocking down γCaMKII in interneurons of the stratum radiatum also leads to disruption of LTPE→I and cognitive function. Ma and colleagues showed that, following learning, hippocampal network oscillations in the gamma and theta bands were significantly weaker in γCaMKII knockout mice than in wild-type mice(He et al. 2021). This finding suggests that impaired experience-dependent oscillations in the hippocampus of γCaMKII PV-KO mice may lead to cognitive dysfunction. In this respect, it will be intriguing to investigate the network oscillation after learning in our condition in future studies.
In the hippocampus, GABAergic local circuit inhibitory interneurons make up approximately 10-15% of the total neuronal cell population (Bezaire & Soltesz 2013). However, these interneurons are diverse in their subtypes, morphology, distribution, and functions (Pelkey et al. 2017; Booker & Vida 2018). In our study, we mainly focused on a subpopulation of interneurons in the stratum radiatum of the hippocampus. Although it is unclear which type of interneuron was recorded in our study, our study indicated that most interneurons in the stratum radiatum do not express CP-AMPARs but express an abundance of NMDARs. These findings are in line with a previous study conducted by Lasmsa et al. (Lamsa et al. 2007). In our study, we observed that approximately 80% of interneurons in the stratum radium were able to induce LTP successfully. This finding contrasts with the observation made by Lamsa et al., who reported a figure of approximately 52% interneurons capable of inducing LTP. The reason for this discrepancy could be attributed to differences in the induction protocol used in the respective studies.
Our study corroborates earlier research that suggests that distinct synaptic mechanisms are involved in LTP induction in the CA1 region of the hippocampus across different subregions (Le Duigou et al. 2015; Lamsa et al. 2007; Kullmann & Lamsa 2011). However, the major breakthrough of our study is the demonstration that astrocytic function serves as the gating mechanism for LTP E→I induction in the stratum radiatum. Additionally, our data reveal that the activation of astrocytes via the Gq-DREADD pathway produces de novo long-lasting potentiation of EPSP in stratum radiatum interneurons and that the knockdown of γCaMKII disrupts cognitive function. These results shed light on the complex mechanisms underlying learning and memory in the hippocampus and may have implications for developing new therapies targeted at modulating astrocytic function for the treatment of memory disorders.
Materials and Methods
Animals
Our study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals and was approved by the ethics committee of Hangzhou City University (registration number: 22061). C57BL/6 male mice (2-4 months) were purchased from Hangzhou Ziyuan Laboratory Animal Corporation and housed in groups of three to four per cage. The mice were maintained on a 12-hour light/dark cycle and were provided with ad libitum access to food and water.
Stereotactic virus injection
Stereotactic virus injection was conducted as described previously (Shen et al. 2021; Shen et al. 2022). Briefly, adult mice were deeply anesthetized with sodium pentobarbital (50 mg/kg) and secured in a stereotaxic device with ear bars (RWD, 68930), while their body temperature was maintained at approximately 37 °C using a heating blanket. Their hair was removed using a razor, and the skin was sterilized with iodophor. A 1-cm incision in the midline was made using sterile scissors. Small burr holes were drilled bilaterally using an electric hand drill at the following coordinates: anteroposterior (AP), 2.3 mm from bregma; mediolateral (ML), ±1.4 mm. Virus particles were then injected bilaterally into the stratum radiatum (1.2 mm from the pial surface) using glass pipettes connected to an injection pump (RWD, R480). The injection rate was controlled at 1 nl/s using the pump. To allow the virus to disseminate into the tissue, a glass pipette was left in place for 10 minutes after each injection. After the injection, the pipettes were gradually removed, and the wound was sutured. For hippocampal interneuron physiological recording, 200 nl AAV2/9 mDLx EGFP (6.4 × 1011 gc/ml) was injected. For hippocampal slice Ca2+ imaging, 500 nl AAV2/5 GfaABC1D GCaMP6f (1.2 × 1012 gc/ml) was injected alone or mixed with 500 nl AAV2/5 GfaABC1D hM3D (Gq) mCherry (5.3 × 1012 gc/ml). To knockdown γCaMKII in hippocampal interneurons, a shRNA sequence (5’-GCAGCTTGCATCGCCTATATC-3’) was used. A total of 200 nl of AAV2/9 mDLx γCaMKII shRNA (6.4 × 1012 gc/ml) was injected. All viruses were generated by Brainvta and Sunbio Medical Biotechnology (Wuhan, https://www.brainvta.tech/ and Shanghai, http://www.sbo-bio.com.cn/). Two to three weeks after viral injection, the mice were utilized for subsequent experiments.
Electrophysiology
Mice were anesthetized with isoflurane, and their brains were quickly extracted and immersed in an ice-cold solution which containing (in mM) 235 sucrose, 1.25 NaH2PO4, 2.5 KCl, 0.5 CaCl2, 7 MgCl2, 20 glucose, 26 NaHCO3, and 5 pyruvate (pH 7.3, 310 mOsm, saturated with 95% O2 and 5% CO2) at 10-11:00 (UTC[+[08:00) in the morning. Coronal hippocampal slices (300-350 μm) were prepared with a vibrating slicer (Leica, V T1200) and incubated for 30-40 min at 32 °C in artificial cerebrospinal fluid (ACSF) containing (in mM) 26 NaHCO3, 2.5 KCl, 126 NaCl, 20 D-glucose, 1 sodium pyruvate, 1.25 NaH2PO4, 2 CaCl2 and 1 MgCl2 (pH 7.4, 310 mOsm, saturated with 95% O2 and 5% CO2).
The slices were transferred to an immersed chamber and continuously perfused with oxygen-saturated ACSF and GABA receptor blockers, picrotoxin (100 μM) and CGP55845 (5 µM), at a rate of 3 ml/min. Interneurons in the dorsal hippocampus of the stratum radiatum or stratum oriens were visualized using infrared differential interference contrast and epifluorescence imaging. Perforated-path recordings were conducted as previously described (Liu et al. 2017). Briefly, perforated whole-cell recordings from stratum radiatum or stratum oriens interneurons were made with pipettes filled with solution containing (in mM) 136 K-gluconate, 9 NaCl, 17.5 KCl, 1 MgCl2, 10 HEPES, 0.2 EGTA, 25 μM Alexa 488, amphotericin B (0.5 mg ml−1) and small amounts of glass beads (5–15 μm in diameter; Polysciences, Inc., Warminster, PA, USA) (pH 7.3, 290 mOsm). The patched neuron was intermittently imaged with epifluorescence to monitor dye penetration. If the patch ruptured spontaneously, the experiment was discontinued.
Whole-cell voltage-clamp recordings were made from either stratum radiatum or stratum oriens interneurons using pipettes with resistance of 3-4 MΩ and filled with a solution containing (in mM) 4 ATP-Na2, 0.4 GTP-Na, 125 CsMeSO3, 10 EGTA, 10 HEPES, 5 4-AP, 8 TEA-Cl, 1 MgCl2, 1 CaCl2 (pH 7.3–7.4, 280–290 mOsm).
Whole-cell current-clamp recordings were made from stratum interneurons using pipettes with a resistance of 4-6 MΩ and filled with a solution containing (in mM) 125 K-Gluconate, 2 MgCl2, 10 HEPES, 0.4 Na+-GTP, 4 ATP-Na2, 10 Phosphocreatine disodium salt, 10 KCl, 0.5 EGTA (pH 7.3–7.4, 280–290 mOsm).
Whole-cell recordings were performed on stratum radiatum astrocytes using pipettes with a resistance of 8-10 MΩ and filled with an intracellular solution containing (in mM) 130 K-Gluconate, 20 HEPES, 3 ATP-Na2, 10 D-Glucose, 1 MgCl2, 0.2 EGTA (pH 7.3-7.4, 280-290 mOsm). In a subset of experiments, 0.14 mM CaCl2 and 0.45 mM EGTA were included in the upper intracellular solution to maintain a stable level of astrocytic concentration (calculation by Web-MaxChelator) (Shen et al. 2022; Henneberger et al. 2010). Astrocytes were identified as described previously (Shen et al. 2021; Shen et al. 2022).
Electrical stimuli were delivered via theta glass pipettes in the Schaffer Collateral of the stratum radiatum, with a 30 s intertrial interval during baseline (10 min) and after LTP induction (45 min). Evoked EPSPs were recorded in the current-clamp model at the resting membrane potential of the stratum radiatum interneuron. After a 10-min stable baseline period, LTP was induced by applying theta-burst stimulation [TBS; five bursts at 200-ms intervals (5 Hz), each burst consisting of five pulses at 100 Hz]. Each burst was paired with 60-ms long depolarizing steps to -10 mV. Six episodes of TBS paired with depolarization were given at 20-s intervals. In the stratum oriens, LTP was induced by applying TBS paired with five 60-ms long hyperpolarizing steps to -90 mV.
sEPSCs were recorded in a whole-cell model at -70 mV in the presence of picrotoxin (100 μM) and D-AP5 (50 μM). Paired pulses were delivered at an interpulse intervals of 50 ms, and the paired-pulse ratio was calculated by dividing the peak amplitude of the second EPSC by the peak amplitude of the first EPSC. To analyze action potential properties, interneurons were recorded at rest and depolarized with 500-ms current injection pulses at 10-pA increments.
To avoid NMDAR-mediated signaling rapidly washing out when recording interneurons in whole-cell mode, electrical stimuli were delivered with a 5 s intertrial interval to measure the I-V relationship for NMDAR-mediated EPSPs within 5 minutes of breaking in. The NMDA/AMPA ratio in the stratum radiatum or stratum oriens interneurons was calculated by measuring the amplitude of NMDAR-mediated EPSCs at +60 mV (50 ms after stimulation) and the peak amplitude of AMPAR-mediated EPSCs recorded at −60 mV. To measure pure NMDAR-mediated EPSCs in interneurons at resting membrane potential in a perforated whole-cell recording model, Mg2+-free ACSF was used, and D-AP5 (50 μM) and picrotoxin (100 μM) were present in ACSF.
Axopatch 700B amplifiers were utilized for patch-clamp recordings (Molecular Devices). The data were filtered at 6 kHz and sampled at 20 kHz before being processed off-line using the pClampfit 10.6 program (Molecular Devices). Bridge balances were automatically compensated for in whole-cell current clamp recordings. Series resistance was not compensated, but negative pulses (-10 mV) were employed to monitor series resistances and membrane resistances. The data were included in the analysis if the series resistances varied by less than 20% over the course of the trial. All experiments were carried out at 32 °C.
Behavioral assays
For the contextual and cued fear conditioning test, the mouse was habituated to the environment and handled for three consecutive days. On the fourth day, the mice were allowed to explore the conditioning cage for 2 min, after which they received three moderate tone-shock pairs [30 stones (80 dB, 4 kHz) coterminating with a foot shock (0.4 mA, 2 s)]. Following conditioning, the mice were returned to their home cages. The next day, they were placed in the same conditioning cage but without receiving any foot shocks, and their freezing behavior was analyzed for the first 5 min. Four hours after the contextual fear conditioning test, the mice were placed in a novel cage with a different shape and texture of the floors compared to those of the conditioning cage. They were allowed to freely explore the new environment for 2 min and then subjected to 3-tone stimulations [(80 dB, 4 kHz) lasting for 30 s] separated by intervals of 90 s, but without receiving any foot-shocks. Once the final tone had ended, all of the mice were allowed to freely explore the chamber for an additional 90 s. Fear responses were measured by calculating the freezing values of the mice using Packwin software (Panlab, Harvard Apparatus, USA). All apparatuses were carefully cleaned with 30% ethanol in between tests.
Immunohistochemistry
Immunohistochemistry was conducted as described previously (Shen et al. 2017; Nikolic et al. 2018; Shen et al. 2021). Briefly, mice were administered a single intraperitoneal injection of 50 mg/kg sodium pentobarbital for anesthesia and were transcardially perfused with phosphate-buffered saline (PBS). After the liver and lungs had become bloodless, the mice were perfused with 4% paraformaldehyde (PFA) in 0.1 M PBS. The brains were quickly removed and placed in 4% PFA at 4 [overnight. Next, the tissues were cryoprotected in successive concentrations of 10%, 20%, and 30% sucrose before being sliced into 20 μm sections using a freezing microtome. After being washed multiple times with PBS, the sections were blocked for 1.5 hours at room temperature (22-24 [) in a blocking solution consisting of 5% bovine serum albumin (BSA) and 1% Triton X-100. Following the blocking step, the sections were incubated with primary antibodies overnight at 4 [. The following antibodies were used: mouse monoclonal anti-GFAP (1/1000, Cell Signaling Technology Cat #3670, RRID: AB_561049), mouse monoclonal anti-NeuN (1/500, Millipore Cat# MAB377, RRID: AB_2298772), and mouse monoclonal anti-GAD67 (1/500, Synaptic Systems Cat# 198 006, RRID: AB_2713980). After washing several times in PBS, the sections were then incubated with the following secondary antibody: Alexa Fluor 594 goat anti-mouse (1/1000, Cell Signaling Technology Cat# 8890, RRID: AB_2714182) at room temperature for 2 h. Afterward, the sections were rinsed several times in PBS and incubated with DAPI for 5 minutes at room temperature. Next, the sections were rinsed again and mounted with Vectashield mounting medium. Images were examined using a confocal laser scanning microscope (Olympus, VT1000) and analyzed using ImageJ (NIH, RRID: SCR_003070).
Ca2+ imaging
Ca2+ signals in hippocampal astrocytes were observed under a confocal microscope (Fluoview 1000; Olympus) with a 40x water immersion objective lens (NA = 0.8). GCaMP6f was excited at 470 nm and the emission signals were further filtered through a 490-582 nm bandpass filter. mCherry was excited at 594 nm and the emission signals were further filtered through a 580-737 nm bandpass filter. Astrocytes located in the hippocampal CA1 region and at least 40 µm away from the slice surface were selected for imaging. Images were acquired at 1 frame per 629 ms. Hippocampal slices were maintained in ACSF containing picrotoxin (100 μM) and CGP55845 (5 µM) using a perfusion system. One episode of TBS was used to stimulate the neuron. CNO (5 μM) was bath-applied to activate hM3D(Gq)-mediated Ca2+ signals. The Ca2+ signal analysis was described previously (Nikolic et al. 2018; Shen et al. 2021; Shen et al. 2022; Shen et al. 2017). In some experiments, image stacks comprising 10-15 optical sections with 1 µM z-spacing were acquired to facilitate the identification of GCaMP6f and hM3Dq (mCherry) coexpression in astrocytes. Briefly, movies were registered using the StackReg plugin of ImageJ to eliminate any x-y drift. Ca2+ signals were then analyzed in selected ROIs using the Time Series Analyzer V3 plugin of ImageJ. GCaMP6f fluorescence was calculated as ΔF/F = (F-F0)/F0. The mean ΔF/F of Ca2+ signals wasanalyzed using Clampfit 10.6.
Statistics
All data processing, figure generation, layout, and statistical analysis were performed using Clampfit 10.6, Prism, MATALAB and Coreldraw. To assess the normality of the data, the Shapiro-Wilk test was used. If the results of the test were not statistically significant (p> 0.05), the data were assumed to follow a normal distribution, and a paired t-test was employed. On the other hand, if the test was statistically significant, a Wilcoxon Signed Rank Test or Mann-Whitney Ran Sum Test was utilized. To statistically analyze cumulative frequency distributions, the Kolmogorov-Smirnov test was employed. When comparing two groups, either a Wilcoxon signed-rank test or a Student’s t-test (paired or unpaired) was used. When comparing three groups, One-way repeated measures (RM) ANOVA followed by Dunnett’s post-hoc test was used.
All values are presented as the mean ± standard error of the mean (SEM). Data were considered significantly different when the p value was less than 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001). The numbers of cells or mice used in each analysis are indicated in the figure legends.
Key Resources Table
Conflict of interest
The authors declare no competing interests.
Data availability statement
Source Data for all figures is available.
Figure supplement
Figure 1—source data 1
LTPE → I in the stratum radiatum is dependent on the activation of NMDA receptors and astrocytic metabolism.
Figure 1—figure supplement 1—source data 1
EGFP is expressed in GABAergic interneurons in the stratum radiatum of the hippocampus.
Figure 1—figure supplement 2—source data 1
EGFP+ interneurons have normal sEPSCs, excitability and PPR.
Figure 1—figure supplement 3—source data 1
Stratum radiatum interneurons show linear rectifying AMPARs and a large NMDAR-mediated component.
Figure 1—figure supplement 4—source data 1
LTPE→I in the stratum oriens is not dependent on astrocytic metabolism or the activation of NMDA receptors.
Figure 1—figure supplement 5—source data 1
Stratum oriens interneurons show inwardly rectifying AMPARs and a negligible NMDAR-mediated component.
Figure 2—source data 1
Astrocyte Ca2+ is involved in the induction of LTP.
Figure 3—source data 1
Activation of astrocytic CB1 receptors causes an increase in astrocytic Ca2+ signals and is involved in the induction of LTPE→I.
Figure 3—figure supplement 1—source data 1
GCaMP6f was expressed in astrocytes.
Figure 4—source data 1
D-Serine release from astrocytes potentiates NMDAR-mediated responses.
Figure 5—source data 1
Astrocytic activation induces de novo LTPE→I.
Figure 5—figure supplement 1—source data 1
hM3Dq was expressed in astrocytes.
Figure 6—source data 1
Impaired hippocampus-dependent long-term memory in γCaMKII knockdown mice.
Figure 6—figure supplement 1—source data 1
Knockdown of γCaMKII in interneurons has no effect on sEPSCs, excitability or the PPR.