Transient Electrical Coupling Regulates Formation of Neuronal Networks
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BRAIN RESEARCH 1129 (2007) 63– 71 available at www.sciencedirect.com www.elsevier.com/locate/brainres Research Report Transient electrical coupling regulates formation of neuronal networks Theresa M. Szaboa, Mark J. Zoranb,⁎ aDominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461, USA bDepartment of Biology, Texas A&M University, MS 3258, Department of Biology, 231 BSBW, College Station, TX 77843-3258, USA ARTICLE INFO ABSTRACT Article history: Electrical synapses are abundant before and during developmental windows of intense Accepted 25 September 2006 chemical synapse formation, and might therefore contribute to the establishment of Available online 6 December 2006 neuronal networks. Transient electrical coupling develops and is then eliminated between regenerating Helisoma motoneurons 110 and 19 during a period of 48–72 h in vivo and in vitro Keywords: following nerve injury. An inverse relationship exists between electrical coupling and Electrical synapse chemical synaptic transmission at these synapses, such that the decline in electrical coupling Chemical synapse is coincident with the emergence of cholinergic synaptic transmission. In this study, we have Helisoma generated two- and three-cell neuronal networks to test whether predicted synaptogenic Synaptogenesis, network capabilities were affected by previous synaptic interactions. Electrophysiological analyses Synapse formation demonstrated that synapses formed in three-cell neuronal networks were not those predicted based on synaptogenic outcomes in two-cell networks. Thus, new electrical and chemical synapse formation within a neuronal network is dependent on existing connectivity of that network. In addition, new contacts formed with established networks have little impact on these existing connections. These results suggest that network- dependent mechanisms, particularly those mediated by gap junctional coupling, regulate synapse formation within simple neural networks. © 2006 Elsevier B.V. All rights reserved. 1. Introduction contacting cells (Kandler and Katz, 1998). In many mammalian brain regions, electrical synapses are abundant before periods Electrical coupling is widespread in invertebrate nervous of chemical synapse formation and then decline in number as systems as well as in the developing mammalian brain. the extent of chemical neurotransmission increases (Bennett Neurons functionally coupled by electrical synapses possess and Zukin, 2004). The appearance of this gap junctional coordinated patterns of spontaneous activity and changes in communication, and the resulting synchronization of neuro- intracellular calcium concentration (Yuste et al., 1992). Some nal activities, is thought to be important for the development of these patterned activities are due to the presence of gap of neuronal assemblies (Kandler and Katz, 1995) and matura- junctions, i.e., membrane channels that provide a conduit for tion of neuronal networks (Allen and Warner, 1991; Walton the passage of electrical current (charged ions) and small and Navarrete, 1991; Roerig and Feller, 2000; Peinado, 2001; cytoplasmic messengers, such as IP3 and cAMP, between Personius et al., 2001; Montoro and Yuste, 2004). For example, ⁎ Corresponding author. Fax: +1 979 845 2891. E-mail addresses: [email protected] (T.M. Szabo), [email protected] (M.J. Zoran). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.09.112 64 BRAIN RESEARCH 1129 (2007) 63– 71 dye coupling between magnocellular neurons of the rat 2004) also occurred in neurite-bearing networks. Cells were hypothalamus increases in vivo during postnatal weeks 1 plated in each possible two-cell configuration, as 19–19, 19–110 and 2 and then decreases during a period of intense chemical and 110–110 partners, and were then incubated for 1 or 5 days synapse formation at postnatal weeks 3 and 4 (Arumugam of contact. Neurite extension in these cultures was elaborate et al., 2005). Chronic block of NMDA receptors significantly and although all somata were initially plated in contact, in reduces uncoupling among these hypothalamic neurons, some cases neuritic fascicles formed between somata sepa- demonstrating that chemical synaptic signaling can regulate rated during process outgrowth (Figs. 1A and B). the developmental uncoupling of gap junctions. Similarly, in spinal motoneurons, NMDA signaling mediates gap junction 2.1. Electrical and chemical neurotransmission in two-cell uncoupling (Mentis et al., 2002). A rapid developmental neuronal networks transition from gap junction-coupled to NMDA receptor- mediated synaptic transmission is also thought to function Electrical connections formed in a manner similar to that seen in cortical network formation (Dupont et al., 2006). previously in vivo (Szabo et al., 2004). After 24 h of cell–cell A sequential progression from electrical coupling to contact, 19–19 and 19–110 pairs possessed coupling coeffi- chemical synaptic transmission exists at regenerating cients (ECCs) of 0.32±0.06 (n=29) and 0.33±0.06 (n=32), res- synapses in the snail, Helisoma, both in vivo and in vitro.At pectively. Two-cell 110–110 networks had an ECC of 0.11±0.05 a period of transition from electrical to chemical commu- (n=6). Following 5 days of contact, 19–19 and 110–110 pairs nication, the two types of synaptic transmission appear to had strong electrical connections (Fig. 1C) while 19–110 pairs be inversely related; i.e., gap junctional coupling is sustained exhibited a reduction in coupling over this same period. Thus, when cholinergic neurotransmission is blocked with tubo- curare and chemical synaptic transmission is increased when electrical coupling is reduced (Szabo et al., 2004). While many examples of the temporal progression from electrical to chemical neurotransmission have been reported (Kandler and Katz, 1998; Szabo et al., 2004; Arumugam et al., 2005; Dupont et al., 2006) and underlying mechanisms have been proposed (Arumugam et al., 2005; Neunuebel and Zoran, 2005), much is still unknown about the influence of these interactions on the formation of neuronal networks. To directly address this question, we have used the defined synapses formed between identified Helisoma neurons in vitro to determine if the process of transient electrical coupling affects subsequent synapse formation in three-cell neuronal networks. Transient electrical synaptic connections develop between Helisoma motoneurons 110 and 19, forming and then disap- pearing over a 48- to 72-h period (Szabo et al., 2004; Neunuebel and Zoran, 2005). The decline in electrical coupling is coincident with the emergence of unidirectional cholinergic synaptic transmission between presynaptic neuron 110 and postsynaptic neuron 19. In the current study, we have generated two- and three-cell neuronal networks to deter- Fig. 1 – Formation of electrical synaptic connections between mine (1) whether new synaptic contacts impact extant neurons paired in outgrowth-permissive conditions. network connectivity, and (2) whether the formation of new (A) Neurons were plated in contacting pairs for 5 days on connections is affected by a neuron's prior synaptic connec- PLL-coated dishes, which promoted adherence to the dish tivity. Our results demonstrate that electrical and chemical surface and outgrowth of processes. After 5 days of synapses form in three-cell networks in a manner not outgrowth, neurons 19 (larger soma) and 110 exhibited predicted from two-cell networks, suggesting that gap junc- extensive neuritic processes. (B) Although neurons were tional coupling and uncoupling plays an important role in plated with contacting somata, in some pairs somata moved regulating neuronal network formation in a cell-specific apart as a large fascicle of processes developed between the manner. two cell bodies. (C) Electrical coupling coefficients (ECCs) were determined as the ratio of postsynaptic to presynaptic voltage changes. ECC values (normalized mean±SEM) for 2. Results 19–19, 110–110, and 19–110 pairs, with 5-day ECC values normalized to 1-day values (19–19: 1-day ECC=0.32±0.06, Buccal neurons 19 and 110 were cultured in conditions n=29 vs. 5-day ECC=0.32±0.07, n=28; NSD (no significant permitting neurite outgrowth, which assured contact among difference). 110–110: 1-day ECC=0.11±0.05, n=6 vs. 5-day neurons of two- and three-cell networks. We first determined ECC=0.15±0.05, n=6; NSD. 19–110: 1-day ECC=0.33±0.06, whether the sequence of synaptogenic events previously n=32 vs. 5-day ECC=0.20±0.05, n=19; *, p<0.05, Student's observed in these neurons lacking neurites (Szabo et al., t-test). BRAIN RESEARCH 1129 (2007) 63– 71 65 electrical connections were maintained or strengthened at significantly in strength (Fig. 2A). A negative correlation homotypic contacts, while heterotypic connections exhibited between these two modes of synaptic communication was significant uncoupling. In addition, by day 5 of contact, an observed for these 110–19 synapses (Fig. 2B). increasing percentage of 110–19 pairs possessed chemical connectivity and PSPs of greater amplitude (Fig. 2A). Choli- 2.2. Formation of three-cell networks nergic synaptic transmission between presynaptic 110 and postsynaptic 19 was present in 50% of cell pairs at day 2 (n=8) Having described the temporal sequence of synapse forma- and 86% of pairs at day 4 (n=7). Chemical neurotransmission tion in two-cell configurations, we tested whether the was not detected in 19–19 (day 2, n=17;