Nicotine Elicits Prolonged Calcium Signaling along Ventral Hippocampal Axons

Citation: Zhong C, Talmage DA, Role LW ( Nicotine Elicits Prolonged Calcium Signaling along Ventral Hippocampal Axons Chongbo Zhong 0 David A. Talmage 0 Lorna W. Role 0 Keiko Abe, The University of Tokyo, Japan 0 1 Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, New York, United States of America, 2 Center for Nervous System Disorder, State University of New York at Stony Brook, Stony Brook, New York, United States of America, 3 Department of Pharmacological Science, State University of New York at Stony Brook, Stony Brook, New York, United States of America, 4 Neuroscience Institute, State University of New York at Stony Brook , Stony Brook, New York , United States of America Presynaptic nicotinic acetylcholine receptors (nAChRs) have long been implicated in the modulation of CNS circuits. We previously reported that brief exposure to low concentrations of nicotine induced sustained potentiation of glutamatergic transmission at ventral hippocampal (vHipp)-striatal synapses. Here, we exploited nAChR subtypeselective antagonists and agonists and 7*nAChR knockout mutant mice (7-/-) to elucidate the signaling mechanisms underlying nAChR-mediated modulation of synaptic transmission. Using a combination of micro-slices culture from WT and 7-/-mice, calcium imaging, and immuno-histochemical techniques, we found that nicotine elicits localized and oscillatory increases in intracellular Ca2+ along vHipp axons that persists for up to 30 minutes. The sustained phase of the nicotine-induced Ca2+ response was blocked by -BgTx but not by DHE and was mimicked by 7*nAChR agonists but not by non-7*nAChR agonists. In vHipp slices from 7-/- mice, nicotine elicited only transient increases of axonal Ca2+ signals and did not activate CaMKII. The sustained phase of the nicotineinduced Ca2+ response required localized activation of CaMKII, phospholipase C, and IP3 receptor mediated Ca2+induced Ca2+ release (CICR). In conclusion, activation of presynaptic nAChRs by nicotine elicits Ca2+ influx into the presynaptic axons, the sustained phase of the nicotine-induced Ca2+ response requires that axonal 7*nAChR activate a downstream signaling network in the vHipp axons. - Funding: This work was funded by a grant from the NIH (NS22061) and a NARSAD Distinguished Investigator Award to LWR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Neuronal nicotinic acetylcholine receptors (nAChRs) influence the excitability of circuits that underlie fundamental aspects of behaviors related to memory, motivation and mood [1-6]. Dysregulation of central nicotinic signaling is linked to devastating neurodegenerative and neuropsychiatric disorders including schizophrenia, Alzheimers disease, depression, anxiety and drug addiction [7-12]. Neuronal nAChRs have been proposed as potential therapeutic targets for cognitive dysfunctions associated with Alzheimers disease and schizophrenia [1315]. Functional nAChRs exist as heteromeric pentamers, comprised of combinations of (2-6) and (2-4) subunits, or homomeric pentamers, comprised of (79) subunits [3,16,17]. The most abundant nAChRs in the brain are 7containing (7*) and 42-containing (42)* subtypes with distinct biophysical and pharmacological properties [18]. Previous studies have shown that (42)* and 7* nAChRs are localized in various cellular domains, including cell bodies, presynaptic terminals, post- and peri-synaptic sites [1921]. Electrophysiological, immunochemical and pharmacological evidence support the presence of (42)* and 7*nAChRs on presynaptic glutamatergic axon terminals, where they modulate the strength of glutamatergic neurotransmission [19,22-25]. Modulation of the release of neurotransmitters (including glutamate, GABA, ACh, and dopamine) by activation of presynaptic nAChRs is the most prevalent mechanism of nicotinic facilitation of synaptic transmission in the CNS [22,23,26,27]. Although nicotinic modulation of circuit excitability by activation of presynaptic nAChRs is critical to CNS function [28-32], the mechanisms by which nAChR activation leads to long-term changes in presynaptic function are not known. We previously reported that brief exposure to low concentrations of nicotine induced sustained (>30min) potentiation of glutamatergic transmission at ventral hippocampal-striatal synapses [33]. Here, we have exploited nAChR subtype-selective antagonists and agonists and 7*nAChRs knockout mutant mice to elucidate the presynaptic cellular mechanisms underlying the nAChR-mediated sustained synaptic potentiation. Materials and Methods vHipp micro-slices cultures and vHipp-nAcc synaptic co-cultures All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 2012) and studies were approved by Institutional Animal Care and Use for Research Committees at Stony Brook University (#1618 and #1792). The cultures were prepared as described previously [33]. Briefly, for vHipp micro-slices cultures, the region of ventral CA1 and subiculum from a single WT or 7 -/mouse (postnatal day 0-3, P0-P3) were dissected, further sliced into 150150 m pieces, and then plated onto poly-Dlysine/laminin-coated glass coverslips (BD Sciences, Bedford, MA) in a minimal volume (50 l) of culture media (Neurobasal, 2% B-27 (GIBCO, Grand Island, NY) and 20 ng/ml brainderived neurotrophic factor (R&D Systems, Minneapolis, MN)) to facilitate attachment. After the microslices settled (1-3 hours at 37C), 100 l of culture media was added. For vHipp-nAcc synaptic co-cultures, nucleus accumbens (nAcc) neurons (ED18 P1) from WT mice (C57BL/6J) were dispersed with 0.25% trypsin (GIBCO, Grand Island, NY) for 15 min at 37C, followed by gentle trituration in culture media. Dispersed nAcc neurons were added to the vHipp microslices plated the prior day at 0.25 ml/coverslip. Cultures were maintained in a humidified 37C, 5% CO2 incubator. To ensure the projections we analyzed were from vHipp, in some experiments, the vHipp microslices were prepared from GFP-reporter transgenic mice. With this co-culture system, we have found that projections from vHipp microslices can make glutamatergic synapses with dispersed nAcc neurons as presynaptic axons [33]. In this study, we used vHipp micro-slices culture alone as presynaptic axons for most of the calcium imaging and immunostaining experiments. Immunostaining and Fluorescent Visualization For standard immuno-detection, cultures were fixed in 4% paraformaldehyde/4% sucrose /PBS (20 min, Room temperature), permeabilized with 0.25% Triton X-100/ PBS (5 min, RT), blocked with 10% normal donkey serum in PBS (30 min, RT), and then incubated in primary antibodies overnight at 4C. The following primary antibodi (...truncated)