Correction NEUROSCIENCE Correction for “Influence of spatially segregated IP3-producing pathways on spike generation and transmitter release in Purkinje cell axons,” by Laura C. Gomez, Shin-ya Kawaguchi, Thibault Collin, Abdelali Jalil, Maria del Pilar Gomez, Enrico Nasi, Alain Marty, and Isabel Llano, which was first published May 1, 2020; 10.1073/pnas.2000148117 (Proc. Natl. Acad. Sci. U.S.A. 117, 11097–11108). The authors note that the complete name for the compound MRS 2365 appeared incorrectly. On page 11107, right column, first full paragraph, lines 6–9,thecompletenameforMRS 2365 should instead appear as “[[(1R,2R,3S,4R,5S)-4-[6-amino-2- (methylthio)-9H-purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hex-1-yl] methyl] diphosphoric acid mono este trisodium salt.” Published under the PNAS license. First published June 1, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2009211117 13176 | PNAS | June 9, 2020 | vol. 117 | no. 23 www.pnas.org Downloaded by guest on September 29, 2021 Influence of spatially segregated IP3-producing pathways on spike generation and transmitter release in Purkinje cell axons Laura C. Gomeza,b, Shin-ya Kawaguchic, Thibault Collina, Abdelali Jalila, Maria del Pilar Gomeza,d, Enrico Nasia,e, Alain Martya, and Isabel Llanoa,1 aSaints Pères Paris Institute for the Neurosciences, Université Paris Descartes, 75006 Paris, France; bDepartment of Physics and Astronomy, University of British Columbia, Vancouver V6T1Z1, BC, Canada; cDepartment of Biophysics, Graduate School of Science, Kyoto University, 606-8502 Kyoto, Japan; dDepartamento de Biología, Universidad Nacional de Colombia, Bogotá 111321, Colombia; and eInstituto de Genética, Universidad Nacional de Colombia, Bogotá 111321, Colombia Edited by Kurt G. Beam, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, and approved March 20, 2020 (received for review January 13, 2020) 2+ It has been known for a long time that inositol-trisphosphate (IP3) intracellular Ca stores that would be distinct from RyR- receptors are present in the axon of certain types of mammalian dependent exocytosis. In addition, IP3Rs could have a functional neurons, but their functional role has remained unexplored. Here role outside the terminal region, since recent studies suggest we show that localized photolysis of IP3 induces spatially con- structural and functional differences between various locations of strained calcium rises in Purkinje cell axons. Confocal immunohis- central axons including the axon initial segment (AIS), paranodal tology reveals that the axon initial segment (AIS), as well as regions, branch points, and axon collaterals (11–13). Surprisingly, terminals onto deep cerebellar cells, express specific subtypes of such possible actions of IP3Rs have not been investigated, and it is Gα/q and phospholipase C (PLC) molecules, together with the up- presently unknown whether axons of central neurons possess stream purinergic receptor P2Y1. By contrast, intermediate parts of functional IP3Rs. the axon express another set of Gα/q and PLC molecules, indicating Purkinje cells (PCs) offer an ideal model to study axonal two spatially segregated signaling cascades linked to IP3 generation. IP3Rs. These neurons contain a high level of IP3Rs in addition to This prompted a search for distinct actions of IP3 in different parts of RyRs, and their implication in calcium signaling in the somato- NEUROSCIENCE Purkinje cell axons. In the AIS, we found that local applications of dendritic compartment has been well documented (14, 15, the specific P2Y1R agonist MRS2365 led to calcium elevation, and that IP photolysis led to inhibition of action potential firing. In reviewed in refs. 8 and 16). Furthermore, IP3Rs were observed in 3 PC axons (17), but it is not known whether these receptors are synaptic terminals on deep cerebellar nuclei neurons, we found that 2+ photolysis of both IP and ATP led to GABA release. We propose able to mobilize Ca from the ER. In PCs, action potentials 3 (APs) produce significant Ca2+ elevation in the AIS (18) and in that axonal IP3 receptors can inhibit action potential firing and in- crease neurotransmitter release, and that these effects are likely con- Ranvier nodes (19, 20), as well as in presynaptic terminals of axon – 2+ trolled by purinergic receptors. Altogether our results suggest a rich and collaterals (21 23). These Ca rises are thought to regulate AP initiation (24), AP propagation (19, 20), and short-term synaptic diverse functional role of IP3 receptors in axons of mammalian neurons. plasticity (21, 22). Using local IP3 delivery by focal photolysis of caged IP3, we show that functional IP3Rs are present in the entire calcium | IP3 | cerebellum | excitability | axon Significance n mammalian neurons, the endoplasmic reticulum (ER) is not Irestricted to the somatodendritic compartment, but it extends IP receptors are well-documented regulators of intracellular also in the axon, up to the most distal presynaptic terminals (1). 3 calcium concentration, but their role in axons of central neu- A number of studies have demonstrated the implication of ER- rons remains unexplored. Here we show that IP receptors are associated intracellular Ca2+ stores in axonal signaling and in 3 present throughout the axon of cerebellar Purkinje cells where the control of neurotransmitter release. Spontaneous, local Ca2+ their activation leads to calcium release from intracellular elevations elicited by Ca2+-induced Ca2+ release (CICR) were stores. We reveal distinct, sharply delimited signaling cascades reported in axons of interneurons of cerebellar slices (2, 3) and leading to IP production in the axon initial segment (AIS), in of cultured hippocampal neurons (4) and are thought to partic- 3 the more distal part of the axon, and in synaptic terminals. In ipate in the control of transmitter release. Recently, ER Ca2+ the AIS, IP3-induced calcium rises inhibit action potential gen- levels were shown to regulate transmitter release (5). The ER eration, whereas similar signals in axonal terminals increase membrane usually carries inositol 1,4,5-trisphosphate receptors transmitter release. Metabotropic purinergic receptors appear (IP3Rs) as well as ryanodine receptors (RyRs, reviewed in ref. 6). 2+ to control the IP3-driven cascade. Altogether, the results reveal Whereas RyRs are sensitive to Ca elevation, IP3Rs are acti- potent actions of IP3 in central axons, which are both diverse vated by phospholipase C (PLC)-driven IP3 production (reviewed and spatially constrained. in ref. 7). The PLC-IP3 pathway has been extensively studied in somatodendritic compartments, where it contributes to post- Author contributions: S.-y.K., T.C., M.d.P.G., E.N., A.M., and I.Ll. designed research; L.C.G., synaptic signaling and induces various forms of long-term synaptic S.-y.K., T.C., A.J., M.d.P.G., E.N., A.M., and I.Ll. performed research; L.C.G., S.-y.K., T.C., A.J., plasticity (reviewed in ref. 8). The same pathway is also well M.d.P.G., E.N., A.M., and I.Ll. analyzed data; and A.M. and I.Ll. wrote the paper with documented in the luminal compartment of exocrine acinar cells, assistance from all authors. where it controls electrolyte secretion and exocytosis (9). Since the The authors declare no competing interest. luminal end of exocrine acinar cells bears a deep-running re- This article is a PNAS Direct Submission. semblance to the axonal compartment of neurons, presumably Published under the PNAS license. reflecting a common ancestral design (10), it is plausible that 1To whom correspondence may be addressed. Email: [email protected]. IP3Rs are likewise important for axonal function. If a functional This article contains supporting information online at https://www.pnas.org/lookup/suppl/ PLC-IP3 pathway exists in central presynaptic terminals, it could doi:10.1073/pnas.2000148117/-/DCSupplemental. offer a way to activate neurotransmitter release using presynaptic www.pnas.org/cgi/doi/10.1073/pnas.2000148117 PNAS Latest Articles | 1of12 axon. We further reveal different IP3-producing pathways in dif- in rats and in mice. Comparing Fig. 1C (staining in mouse) to ferent axon regions using immunocytochemistry. Finally, we demon- Fig. 1A (staining in rat) shows that IP3R-I expression in the granule strate diverse consequences of IP3R activation depending on IP3R celllayerwassimilarinratsandinmice.Expressionwasconfined localization: while IP3R activation in terminals leads to neurotrans- to the axons of PCs, as ascertained by colocalization with the well- mitter release, IP3R activation in the AIS inhibits AP firing. known PC marker calbindin (Fig. 1C). Results IP3 Photolysis in PC Axons. To investigate whether axonal IP3Rs PC Axon Staining with IP3R Antibody. To investigate the spatial were functional, we used localized IP3 photolysis, as schematized in Fig. 2A. PCs were dialyzed with a K gluconate-based solu- distribution of IP3R type I, anti-IP3R-I antibodies were tested in sagittal cerebellar slices of both rats and mice at ages ranging tion supplemented with the calcium probe Oregon Green 488 from postnatal day 12 (PN12) to PN45. As illustrated in Fig. 1A BAPTA-1 (OGB1) and caged IP3. Two-photon laser scanning for a rat at PN16 (Left), prominent staining was obtained in the microscopy (2PLSM) was used to follow the PC axon as it somatodendritic compartment of PCs as well as in their axons. courses through the granule cell layer (Fig. 2B). To elicit a local IP R-I staining was present throughout the entire axonal ar- IP3 rise, a short pulse of 405-nm laser excitation was delivered 3 to a diffraction-limited spot in the axon (Methods). Such stimuli borization, including the axonal projections to the deep cere- elicited local Ca responses, as shown in Fig. 2 C and D for two bellar nuclei (DCN), where structures resembling synaptic i different regions of the PC axon illustrated in Fig. 2B. The first D boutons over DCN somata could be visualized (Fig. 1 ). No region is located at an axonal branch point (Fig.