NMDA receptors regulate developmental gap junction uncoupling via CREB signaling Harsha Arumugam 1 , Xinhuai Liu 1 , Paul J Colombo 2 , Roderick A Corriveau 3 & Andrei B Belousov 1 Signaling through gap junctions (electrical synapses) is important in the development of the mammalian central nervous system. Abundant between neurons during postnatal development, gap junction coupling subsequently decreases and remains low in the adult, confined to specific subsets of neurons. Here we report that developmental uncoupling of gap junctions in the rat hypothalamus in vivo and in vitro is associated with a decrease in connexin 36 (Cx36) protein expression. Both developmental gap junction uncoupling and Cx36 downregulation are prevented by the blockade of NMDA glutamate receptors, action potentials and the calcium–cyclic AMP response element binding protein (CREB), and are accelerated by CREB overexpression. Developmental gap junction uncoupling and Cx36 downregulation are not affected by blockade of non-NMDA glutamate receptors, and do not occur in hypothalamic neurons from NMDA receptor subunit 1 (NMDAR1) knockout mice. These results demonstrate that NMDA receptor activity contributes to the developmental uncoupling of gap junctions via CREB-dependent downregulation of Cx36. Gap junctions are widespread in the developing mammalian CNS and have been implicated in many developmental events, including neu- rogenesis 1 , neuronal differentiation 2 , cell death 3 , cell migration 4 , synaptogenesis and neural circuit formation 5–11 . The passage of Ca 2+ , inositol 1,4,5-trisphosphate (IP 3 ), cyclic AMP (cAMP) and other signaling molecules through gap junctions may coordinate metabolic and transcriptional activities in developing neurons 9,12–14 . Gap junc- tions also contribute to a primitive form of synchronized spontaneous activity that is a hallmark of the developing brain 15,16 . This network- driven activity often involves cooperation between gap junctions and chemical synapses such as cholinergic synapses in the retina 9,17,18 , cholinergic and GABA synapses in the spinal cord 19 and GABA synapses in the hippocampus 15,20 . The incidence of gap junction coupling decreases during postnatal development. In the spinal cord 6,21,22 , hippocampus 20 , neocortex 23,24 and striatum 25 of the rat, and the visual cortex of the ferret 26 , uncoupling occurs during postnatal weeks 1 to 3 and thus overlaps with the major period of chemical synapse formation and increased synaptic activity 26 . Here we tested the hypothesis that the maturation of glutamatergic transmission is responsible for developmental gap junction uncoupling. The model system for the present study is the hypothalamus, which expresses gap junctions and is critical for homeostatic regulation and coordination of cardiovascular, nervous and endocrine functions 27 . We demonstrate that NMDA receptor–mediated glutamatergic transmission is required for developmental gap junction uncoupling via Ca 2+ -dependent signal transduction pathways and CREB-dependent regulation of Cx36 expression. RESULTS Developmental gap junction uncoupling in vivo Developmental changes in neuronal gap junction coupling were deter- mined in acute slices of the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the rat hypothalamus. We used the coupling tracer neurobiotin, which passes through gap junctions, and the dextran Alexa Fluor 594, which is gap junction impermeable (Fig. 1a–c). In parallel experiments, we used western blots on dissected hypothalamus to study developmental changes in the expression of Cx36, a gap junction protein that is neuron-specific and essential for functional gap junctions in the hypothalamus 28–30 . The incidence of dye coupling—that is, the percentage of primary-labeled neurons coupled to one or more secondary-labeled neurons—and the expression of Cx36 both increased during the first two weeks of postnatal hypothalamic development (Supplementary Fig. 1 online). This change indicates an initial developmental increase in neuronal gap junction coupling between the time of birth and postnatal day 15 (P15). The incidence of dye coupling and the expression of Cx36 decreased between P15 and P30 (Fig. 1d–f and Table 1), indicating subsequent developmental gap junction uncoupling. Chronic administration in vivo of the NMDA receptor antagonist dizocilpine (MK-801) (0.5–1.5 mg kg 1 sub- cutaneously every 24 h from P1 to P30) suppressed this developmental uncoupling and Cx36 downregulation (Fig. 1d–f and Table 1). The initial increases (from P1 to P15) in gap junction coupling and Cx36 expression were not affected by MK-801 (Fig. 1d–f; saline versus MK- 801 on P15). In contrast to Cx36, levels of Cx43 (a presumptive glial connexin 28 ) increased in the hypothalamus between P15 and P30, and this increase was not affected by chronic MK-801 (Fig. 1g,h). Received 13 July; accepted 28 September; published online 20 November 2005; doi:10.1038/nn1588 1 Department of Cell and Molecular Biology and 2 Department of Psychology, Tulane University, New Orleans, Louisiana 70118, USA. 3 Department of Cell Biology and Anatomy, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA. Correspondence should be addressed to A.B.B. (belousov@tulane.edu). 1720 VOLUME 8 [ NUMBER 12 [ DECEMBER 2005 NATURE NEUROSCIENCE ARTICLES © 2005 Nature Publishing Group http://www.nature.com/natureneuroscience