Long-term potentiation (LTP) of synaptic transmission represents the cellular basis of learning and memory. Astrocytes have been shown to regulate synaptic transmission and plasticity. However, their involvement in specific physiological processes that induce LTP in vivo remains unknown. Here we show that in vivo cholinergic activity evoked by sensory stimulation or electrical stimulation of the septal nucleus increases Ca²⁺ in hippocampal astrocytes and induces LTP of CA3-CA1 synapses, which requires cholinergic muscarinic (mAChR) and metabotropic glutamate receptor (mGluR) activation. Stimulation of cholinergic pathways in hippocampal slices evokes astrocyte Ca²⁺ elevations, postsynaptic depolarizations of CA1 pyramidal neurons, and LTP of transmitter release at single CA3-CA1 synapses. Like in vivo, these effects are mediated by mAChRs, and this cholinergic-induced LTP (c-LTP) also involves mGluR activation. Astrocyte Ca²⁺ elevations and LTP are absent in IP₃R2 knock-out mice. Downregulating astrocyte Ca²⁺ signal by loading astrocytes with BAPTA or GDPβS also prevents LTP, which is restored by simultaneous astrocyte Ca²⁺ uncaging and postsynaptic depolarization. Therefore, cholinergic-induced LTP requires astrocyte Ca²⁺ elevations, which stimulate astrocyte glutamate release that activates mGluRs. The cholinergic-induced LTP results from the temporal coincidence of the postsynaptic activity and the astrocyte Ca²⁺ signal simultaneously evoked by cholinergic activity. Therefore, the astrocyte Ca²⁺ signal is necessary for cholinergic-induced synaptic plasticity, indicating that astrocytes are directly involved in brain storage information. Information processing in the brain was classically thought to rely solely on neurons, whereas astrocytes, the most abundant glial cells in the brain, were considered supportive cells for neurons. However, astrocytes are known to respond to neuronal signals and regulate the function of synapses, so they may indeed serve active roles during information processing and storage in the brain. We investigated whether these phenomena occur in vivo and whether astrocytes participate in synaptic plasticity and long-term potentiation (LTP), which are thought to represent the cellular basis of learning and memory. We found that sensory stimulation in rodents triggers the activity of neurons that release the neurotransmitter acetylcholine and activates astrocytes in the hippocampus, a brain region involved in learning and memory. Acetylcholine elevates intracellular calcium in astrocytes, which then stimulates the release of glutamate, another neurotransmitter. Glutamate released from astrocytes acts on synaptic receptors, increasing synaptic strength and leading to LTP of the efficacy of transmission in synapses. This is the first demonstration of the direct involvement of astrocytes in the generation of in vivo LTP. We suggest that astrocytes are cellular sources of signals underlying synaptic plasticity and are directly involved in memory processes and brain information storage.

Cholinergic–glutamatergic interactions influence forms of synaptic plasticity that are thought to mediate memory and learning. We testedin vitrothe induction of long-lasting synaptic enhancement at Schaffer collaterals by acetylcholine (ACh) at the apical dendrite of CA1 pyramidal neurons andin vivoby stimulation of cholinergic afferents.In vitroACh induced a Ca²⁺wave and synaptic enhancement mediated by insertion of AMPA receptors in spines. Activation of muscarinic ACh receptors (mAChRs) and Ca²⁺release from inositol 1,4,5-trisphosphate (IP₃)-sensitive stores were required for this synaptic enhancement that was insensitive to blockade of NMDA receptors and also triggered by IP₃uncaging. Activation of cholinergic afferentsin vivoinduced an analogous atropine-sensitive synaptic enhancement. We describe a novel form of synaptic enhancement (LTP_IP3) that is inducedin vitroandin vivoby activation of mAChRs. We conclude that Ca²⁺released from postsynaptic endoplasmic reticulum stores is the critical event in the induction of this unique form of long-lasting synaptic enhancement.

Analysis of the cholinergic regulation of glutamatergic neurotransmission is an essential step in understanding the hippocampus because it can influence forms of synaptic plasticity that are thought to underlie learning and memory. We studied in vitro the cholinergic regulation of excitatory postsynaptic currents (EPSCs) evoked in rat CA1 pyramidal neurons by Schaffer collateral (SC) stimulation. Using ‘minimal’ stimulation, which activates one or very few synapses, the cholinergic agonist carbamylcholine (CCh) increased the failure rate of functional more (36 %) than of silent synapses (7 %), without changes in the EPSC amplitude. These effects of CCh were insensitive to manipulations that increased the probability of release, such as paired pulse facilitation, increases in temperature and increases in the extracellular Ca²⁺ : Mg²⁺ ratio. Using ‘conventional’ stimulation, which activates a large number of synapses, CCh inhibited more the pharmacologically isolated non‐NMDA (86 %) than the NMDA (47 %) EPSC. The changes in failure rate, EPSC variance and the increased paired pulse facilitation that paralleled the inhibition imply that CCh decreased release probability. Muscarine had similar effects. The inhibition by both CCh and by muscarine was prevented by atropine. We conclude that CCh reduces the non‐NMDA component of SC EPSCs by selectively inhibiting transmitter release at functional synapses via activation of muscarinic receptors. The results suggest that SCs have two types of terminals, one in functional synapses, selectively sensitive to regulation through activation of muscarinic receptors, and the other in silent synapses less sensitive to that regulation. The specific inhibition of functional synapses would favour activity‐dependent plastic phenomena through NMDA receptors at silent synapses without the activation of non‐NMDA receptors and functional synapses.

We had described a muscarinic-mediated long-term synaptic enhancement at Schaffer collateral synapses caused by the insertion of AMPARs in spines of rat hippocampal CA1 pyramidal neurons that requires Ca²⁺release from IP3-sensitive stores (Fernández de Sevilla et al., 2008). We now show that this AMPA-mediated LTP_IP3is precisely matched by an amplification of NMDAR-mediated transmission. The enhanced AMPAR transmission involves SNARE protein activity and CaMKII activation. The amplification of NMDA transmission requires combined CaMKII, PKC, and SRC kinase activity without detectable surface incorporation of NMDARs, suggesting that changes in receptor properties mediate this process. The enhanced AMPAR- and NMDAR-mediated transmission markedly reduce the induction threshold of “Hebbian” LTP. We conclude that both modes of glutamatergic synaptic potentiation may play a critical functional role in the regulation of the learning machinery of the brain by adding flexibility to the demands of the hippocampal network.

The cellular mechanisms that mediate spike timing–dependent plasticity (STDP) are largely unknown. We studied in vitro in CA1 pyramidal neurons the contribution of AMPA and N-methyl-d-aspartate (NMDA) components of Schaffer collateral (SC) excitatory postsynaptic potentials (EPSPs; EPSP_AMPA and EPSP_NMDA) and of the back-propagating action potential (BAP) to the long-term potentiation (LTP) induced by a STDP protocol that consisted in pairing an EPSP and a BAP. Transient blockade of EPSP_AMPA with 7-nitro-2,3-dioxo-1,4-dihydroquinoxaline-6-carbonitrile (CNQX) during the STDP protocol prevented LTP. Contrastingly LTP was induced under transient inhibition of EPSP_AMPA by combining SC stimulation, an imposed EPSP_AMPA-like depolarization, and BAP or by coupling the EPSP_NMDA evoked under sustained depolarization (approximately −40 mV) and BAP. In Mg²⁺-free solution EPSP_NMDA and BAP also produced LTP. Suppression of EPSP_NMDA or BAP always prevented LTP. Thus activation of NMDA receptors and BAPs are needed but not sufficient because AMPA receptor activation is also obligatory for STDP. However, a transient depolarization of another origin that unblocks NMDA receptors and a BAP may also trigger LTP.

Neocortical cholinergic activity plays a fundamental role in sensory processing and cognitive functions, but the underlying cellular mechanisms are largely unknown. We analyzed the effects of acetylcholine (ACh) on synaptic transmission and cell excitability in rat “barrel cortex” layer V (L5) pyramidal neurons in vitro. ACh through nicotinic and M1 muscarinic receptors enhanced excitatory postsynaptic currents and through nicotinic and M2 muscarinic receptors reduced inhibitory postsynaptic currents. These effects increased excitability and contributed to the generation of Ca²⁺ spikes and bursts of action potentials (APs) when inputs in basal dendrites were stimulated. Ca²⁺ spikes were mediated by activation of NMDA receptors (NMDARs) and L-type voltage-gated Ca²⁺ channels. Additionally, we demonstrate in vivo that basal forebrain stimulation induced an atropine-sensitive increase of L5 AP responses evoked by vibrissa deflection, an effect mainly due to the enhancement of an NMDAR component. Therefore, ACh modified the excitatory/inhibitory balance and switched L5 pyramidal neurons to a bursting mode that caused a potent and sustained response enhancement with possible fundamental consequences for the function of the barrel cortex.