Synaptically Released Acetylcholine Evokes Ca²⁺Elevations in Astrocytes in Hippocampal Slices

Abstract

Recent results have demonstrated the existence of bidirectional communication between glial cells and neurons. We investigated in brain slices whether rat hippocampal astrocytes respond to acetylcholine synaptically released by an extrinsic pathway. We stimulated the stratum oriens/alveus, which contains cholinergic afferents from the septum and diagonal band of Broca, and recorded whole-cell membrane currents and intracellular Ca2+ levels of astrocytes located in the hippocampal stratum oriens. Nerve-fiber stimulation evoked a long-lasting inward current and increased the Ca2+ levels in astrocytes. Both astrocytic responses were abolished by tetrodotoxin or Cd2+ and were increased by 4-aminopyridine, indicating that the responses were attributable to synaptically released neurotransmitter. The inward current was inhibited by glutamate transporter antagonists, indicating that it was attributable to the electrogenic glutamate transporter activity. The synaptically evoked intracellular Ca2+ elevations were not affected by glutamate receptor antagonists but were abolished by atropine, indicating that they were mediated by muscarinic cholinergic receptors. Thapsigargin prevented the Ca2+ elevation but did not modify the inward current, indicating that the Ca2+ signal was attributable to intracellular Ca2+ mobilization. These results indicate that hippocampal astrocytes respond to acetylcholine released by synaptic terminals. The synaptically released acetylcholine acts on muscarinic receptors, mobilizing Ca2+ from the intracellular stores. Different regions in the recorded astrocytes showed independent stimulus-induced Ca2+ variations, suggesting the existence of subcellular domains in the astrocytic responses evoked by the synaptic cholinergic activity. Therefore, our results show the existence of cholinergic neuron-astrocyte signaling and suggest that astrocytes are a target of axonal inputs from different brain areas.

Mammalian motor neurons corelease glutamate and acetylcholine at central synapses

“…(i) They were characterized as being large neurons located in the ventral horn that could be activated antidromically from the ventral root. In accordance with classical criteria, the antidromic spike followed high stimulus frequencies (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) with no jitters in the activation latencies (see Fig. 1b).…”

“…When ACh (100 M) was bath-applied, MEC used in the same concentrations completely blocked the depolarizing response in RCs, whereas CNQX, AP5, and KYN (n ϭ 3) had no effect on the depolarization induced by ACh. Atropine (n ϭ 4; 10 M) had no effect on the d-TC͞MEC-resistant EPSCs, showing that ACh released from MNs is not acting via muscarinic receptors on other cells to release glutamate, as has been shown, for example, in hippocampus (14).…”

Properties of Synaptically Evoked Astrocyte Calcium Signal Reveal Synaptic Information Processing by Astrocytes

“…Slices were then transferred to an immersion recording chamber and superfused with gassed ACSF. In some cases, the ACSF contained 100 M 4-aminopyridine (4-AP) to enhance astrocytic responses (Porter and McCarthy, 1996;Araque et al, 2002). In experiments designed to optimize NMDAR activation, the extracellular Mg 2ϩ was equimolarly substituted by Ca 2ϩ , and 10 M glycine was added.…”

“…This concept is based on the demonstration that astrocytes display a form of excitability based on intracellular Ca 2ϩ variations (Pasti et al, 1997;Verkhratsky et al, 1998;Haydon, 2001;Charles and Giaume, 2002;Haydon and Araque, 2002;Nedergaard et al, 2003), respond to synaptically released neurotransmitters (Porter and McCarthy, 1996;Pasti et al, 1997;Grosche et al, 1999;Latour et al, 2001;Araque et al, 2002), and modulate neuronal excitability and synaptic transmission by releasing neuroactive substances through, at least some of them, Ca 2ϩ -dependent mechanisms (Araque et al, 1998a,b;Kang et al, 1998;Newman and Zahs, 1998;Robitaille, 1998;Parri et al, 2001;Beattie et al, 2002;Brockhaus and Deitmer, 2002;Volterra and Bezzi, 2002;Newman, 2003;Zhang et al, 2003;Fiacco and McCarthy, 2004;Liu et al, 2004).…”

Astrocytes Display Complex and Localized Calcium Responses to Single-Neuron Stimulation in the Hippocampus

“…Indicator dyes with blue excitation and green emission spectra, such as Fluo-3 AM (Dani et al, 1992;Araque et al, 2002), Fluo-4 AM (Simard et al, 2003;Schipke et al, 2008), Calcium Green 1 (Fiacco and McCarthy, 2004), and Oregon Green-BAPTA-1 (Agulhon et al, 2010), are commonly used to detect intracellular calcium elevations. How-ever, these dyes are incompatible with ChR2 because they have overlapping excitation wavelengths.…”