Cholinergic agonists increase intracellular Ca2+ in cultured human microglia

Neuroscience Letters 255 (1998) 33–36

Cholinergic agonists increase intracellular Ca2+ in cultured human microglia Lili Zhang a, James G. McLarnon b, Vikram Goghari b, Yong Beom Lee a, Seung U. Kim a, Charles Krieger a ,* a

Division of Neurology, Department of Medicine, Faculty of Medicine, The University of British Columbia, 2211 Wesbrook Mall, Vancouver, British Columbia V6T 2B5, Canada b Department of Pharmacology and Therapeutics, Faculty of Medicine, The University of British Columbia, 2176 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada Received 9 July 1998; received in revised form 22 August 1998; accepted 25 August 1998

Abstract Microglia are resident phagocytic cells in the central nervous system (CNS), and can be activated in response to various stimuli including neurotransmitters. Using fura-2 imaging, we investigated the effects of carbachol (CCh), a cholinergic agonist, on [Ca2+]i in cultured human microglia. Treatment of microglia with CCh (100 mM) produced a transient increase in [Ca2+]i, which was atropine-sensitive and was associated with release from intracellular Ca2+ stores. Successive applications of CCh showed a change in the amplitude of the [Ca2+]i signal consistent with desensitization. These results show that human microglia express functional muscarinic receptors and respond to cholinergic agonists. The rapid change of [Ca2+]i in microglia may serve as a second messenger to trigger downstream cascades which contribute to signalling pathways in CNS pathology.  1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Microglia; Intracellular free calcium; Fura-2; Carbachol; Acetylcholine; Human

Microglia are resident phagocytic cells in the central nervous system (CNS) and serve as scavenger cells in the event of infection, inflammation, trauma, ischemia and neurodegeneration [8]. In the latter case microglia are associated with the amyloid deposits found in senile plaques in the brains of patients with Alzheimer’s disease (AD) and may also be involved in the production of amyloid precursor protein (APP). The functions of microglia in the formation of plaques is presently unclear but in some cases the cells are observed in association with synaptic structures [7]. As cholinergic fibers exhibit synapses around some plaques in patients with AD [5], an interesting possibility is that human microglia could respond to cholinergic agonists, a point investigated in the present work. The procedures used to isolate and identify human microglia have been previous described [6,12]. Enriched cultures * Corresponding author. Tel.: +1 604 8227504; fax: +1 604 8227897.

of human microglia were prepared from fetal brain tissue (12–20 weeks gestation) following therapeutic abortion (legal abortions were performed in an authorised centre and approval for the use of tissues was obtained from the Ethics Committee of the University of British Columbia). The purity of microglia in cultures was near 98%. Fura-2 imaging was performed [2,12] on microglial cells incubated at 37°C for 25 min with 5 mM fura-2 acetoxymethyl ester (fura-2/AM; Molecular Probes, Eugene, OR) plus 0.02% pluronic acid, and 0.02% BSA in a HEPES-buffered Hanks’ balanced salt solution (HBSS, pH 7.4). The latter solution contained (in mM) NaCl, 145; KCl, 2.5; MgCl2, 1.0; CaCl2, 1.8; HEPES, 20; glucose, 10. In calcium-free experiments the CaCl2 was omitted and 50 mM EGTA was added. The coverslips were then transferred to a perfusion chamber and fluorescence was measured using a Nikon 40× fluorite epifluorescence objective fitted to an inverted microscope. The emitted fluorescence was detected by a silicone intensified target (SIT) video camera and fed to a

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00706- X

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computer equipped with image-detection software (ImageI; Universal Imaging) and an analog-to-digital video digitizer board. The ratio of intensities at 340 and 380 nm were measured and converted to calcium using a standard calibration curve [2]. Data were analysed using a spreadsheet (MS Excel) and results shown are the mean ± SEM values. Cultured human microglia exhibited two types of morphology, amoeboid (round) and ramified forms. Microglia having both types of morphology were studied; we did not attempt to distinguish between the [Ca2+]i responses from either type. In the present experiments we studied the [Ca2+]i responses of microglia to CCh in a total of 77 cells and to ACh in a total of 27 cells. Microglia were chosen for study only when a stable baseline level of [Ca2+]i was achieved under control conditions. The resting [Ca2+]i of human microglia was in the range of 50–70 nM when studied in a 1.8 mM Ca2+-containing solution. The application of 100 mM CCh to microglia in 1.8 mM Ca2+-containing HBSS produced a rapid, transient increase in [Ca2+]i. A typical response is shown in Fig. 1A, which represents the average [Ca2+]i of six cells. This transient response had a rise time of less than 1 min and recovered within 2 min. Transient increases in [Ca2+]i were observed in 44 out of 77 cultured microglia. Acetylcholine (100 mM) also produced a transient response in [Ca2+]i as shown in Fig. 1B. Overall, ACh was applied to 27 microglia and 17 of these cells had responses similar to that shown in Fig. 1B. In subsequent studies we used CCh to investigate further the properties of this [Ca2+]i signal. In order to determine if cholinergic stimulation exhibited any desensitization, successive applications of CCh (100 mM) were performed on CCh-responsive microglia. A second application of CCh given 5–6 min after the first application produced a response with an amplitude of approximately 50–60% of the initial [Ca2+]i peak. Fig. 2A shows a representative example of CCh desensitization in cultured microglia, which was observed in another four experiments. This suggests that microglia desensitize to repeated exposures of CCh; we did not investigate if the degree of desensitization was changed with different times between CCh applications. Co-application of carbachol and atropine (10 mM), an inhibitor of muscarinic receptors, did not produce any [Ca2+]i response (data not shown) indicating muscarinic receptor antagonism (n = 42). To determine the source of the CCh-induced [Ca2+]i increase in human microglia, we examined the influence of extracellular Ca2+. After obtaining a stable baseline in 1.8 mM Ca2+-containing HBSS, microglia were exposed to Ca2+-free HBSS for a short period (less than 2 min), then CCh (100 mM) was applied. This CCh application produced a transient [Ca2+]i response which was very similar to that obtained with extracellular Ca2+. After the CCh was washed out for about 5–6 min, a second CCh application in Ca2+-free HBSS gave no response (Fig. 2B). Similar results were found in 21 cells in three independent experiments. These results suggest that the change in [Ca2+]i

resulting from CCh stimulation is largely mediated by release from intracellular Ca2+ stores as a response was observed in Ca2+-free HBSS, and a second CCh application produced no response, presumably due to failure of Ca2+ store refilling in the absence of extracellular Ca2+. The present study is the first demonstration that human microglia express functional muscarinic receptors and respond to cholinergic neurotransmitters with a Ca2+ transient. The CCh-induced calcium response was atropine-sen-

Fig. 1. Effect of cholinergic agonists on [Ca2+]i in cultured human microglia. The average baseline [Ca2+]i of human microglia is about 50–70 nM. (A) Application of muscarinic receptor agonist, CCh (100 mM), produced an increase in [Ca2+]i when in 1.8 mM Ca2+-containing HBSS. Data shown are the mean response calculated from data on six cells. (B) Application of ACh (100 mM) to microglia in 1.8 mM Ca2+-containing HBSS elicited a similar [Ca2+]i transient response. Values shown are the mean response calculated from data on five cells.

L. Zhang et al. / Neuroscience Letters 255 (1998) 33–36

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system to stimulate intracellular Ca2+ store release as reported for muscarinic responses in a variety of other cell types [3]. Activated microglia are observed in neurodegenerative diseases and may be involved in the initiation or progression of CNS pathology. In Alzheimer’s disease, microglia are associated with amyloid plaques and appear in proximity to cholinergic terminals [5,7]. APP is expressed at high levels by neurons, astrocytes, and activated microglia in mammalian brain and the regulation of APP secretion occurs through a complex mechanism that involves activation of various cell surface receptors coupled to protein kinases. It has been previously shown that APP secretion is enhanced with agonists for M1 or M3 muscarinic receptors [1,4]. In addition, phorbol esters, which stimulate protein kinase C (PKC) have also been reported to increase secretion of APP in some cell types [1,4] and are known to increase [Ca2+]i in human microglia [12]. Our results suggest that as human microglia generate a Ca2+ transient in response to carbachol and acetylcholine, it is possible that cholinergic stimulation, PKC activation, and increased [Ca2+]i may be involved in the regulation of APP secretion by microglia. Furthermore, a recent study has reported cultured rat brain microglia and astrocytes are able to synthesize acetylcholine [10] leading to the possibility that glial processing of this agonist may be altered in Alzheimer’s disease. This work was supported by grants from Columbia Health Research Foundation (to Natural Science and Engineering Research Canada (to J.G.M.), and the MS Society of S.U.K).

Fig. 2. Successive applications of CCh (100 mM) show changes in the [Ca2+]i response. (A) Application of CCh in Ca2+-containing HBSS produced an increase in [Ca2+]i. A second application of CCh after 6 min resulted in a second [Ca2+]i response with an amplitude of about 60% of the first response. Data shown are the response from a single cell. (B) In Ca2+-free HBSS, CCh (100 mM) produced a transient Ca2+ increase, but no response was obtained with the second CCh (100 mM) application 6 min later. Values shown are the mean response from data on five cells.

sitive, exhibited desensitization and resulted primarily from release of Ca2+ from intracellular stores since the responses were abolished in the absence of extracellular Ca2+. A CChinduced Ca2+ transient has also been observed in cultured rat microglia [11]. It is important to note that human and rodent microglia may differ in some respects; for example, inducible nitric oxide synthase (iNOS) was found in rodent microglia but was not present in human cells [9]. Our observations suggest that functional muscarinic receptors in microglia may be coupled to the inositol phosphate second messenger

the British C.K.), the Council of Canada (to

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