The regulation and role of neuronal gap junctions during development

Abstract

Coupling of neurons by electrical synapses (gap junctions) transiently increases in the mammalian CNS during development and plays a role in a number of developmental events, including neuronal death. The coupling subsequently decreases and remains low in the adult, confined to specific subsets of neurons. In a recent study we have demonstrated that the developmental increase in neuronal gap junction coupling is regulated by the balance between the activity of two neurotransmitter receptors, group II metabotropic glutamate receptors (mGluR) and GABA_A receptors. Specifically, we found that activation of group II mGluRs induces the developmental increases in neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein) and activation of GABA_A receptors counteracts to these increases. We also established that the regulation by both neurotransmitter receptors is via a neuron-restrictive silencer element in the Cx36 gene promoter and the 3′-untranslated region of the Cx36 mRNA. Importantly, we demonstrated that mechanisms for the developmental increase in neuronal gap junction coupling directly control the death/survival mechanisms in developing neurons.

Transient coupling of large groups of neurons by electrical synapses (gap junctions) is a general phenomenon in the developing mammalian central nervous system (CNS): it has been documented in different regions of the CNS (neocortex, hippocampus, hypothalamus, striatum, locus coeruleus, spinal cord, etc.,) and in different species (rat, mouse, ferret, opossum, etc.).¹ Neuronal gap junction coupling increases during late embryonic and/or early postnatal development and plays an important role in a number of developmental events, including neuronal differentiation, cell death, cell migration, synaptogenesis and neural circuit formation.^2–8 The coupling decreases during later stages of development, which occurs in response to increasing chemical synaptic (glutamatergic) transmission and activation of N-methyl-D-aspartate (NMDA) receptors.⁹

Recently, we characterized the mechanisms that are responsible for the developmental increase in neuronal gap junction coupling and the role of these mechanisms in the regulation of death/survival mechanisms in developing neurons.¹⁰ The experiments were conducted in developing neuronal cultures prepared from the rat and mouse hypothalamus and cortex and in the hypothalamus of developing rats in vivo. We established the role for two neurotransmitter receptors: group II metabotropic glutamate receptors (mGluR) and γ-aminobutyric acid A receptors (GABA_ARs) (Fig. 1). Specifically, using dye coupling, electrotonic coupling, western blots and small interfering RNA, we showed that a prolonged (2 week) activation of group II mGluRs augments, and inactivation prevents, the developmental increases in neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein). However, changes in GABA_A receptor activity had the opposite effects. We also established that the regulation by group II mGluRs is via cAMP/PKA-dependent signaling pathways (that are negatively coupled to the group II mGluRs¹¹) and the regulation by GABA_ARs (which are excitatory during development¹²) is via Ca²⁺ influx through voltage-gated Ca²⁺channels and activation of protein kinase C (Fig. 1). We also showed that other glutamate receptors, acetylcholine receptors and GABA_B receptors are not involved in these regulatory mechanisms. Moreover, agonists and antagonists of group II mGluRs and GABA_ARs did not affect the expression of Cx43 (that is a presumptive glial connexin¹³ and also increases during development⁹), suggesting that the regulation is specific for neuronal, but not glial gap junctions. Moreover, neither developmental nor receptor-dependent increases in neuronal gap junction coupling were observed in Cx36-deficient neurons, suggesting that this regulation is exclusive for Cx36.

Figure 1.

Simplified model of mechanisms for the developmental increase in neuronal gap junction coupling and connexin 36 expression in the mammalian CNS. Note that in developing neurons, the GABA_AR-dependent pathway is excitatory and causes cell depolarization and Ca²⁺ influx. cAMP, cyclic AMP; GABA_AR, GABA_A receptor; IImGlur, group II metabotropic glutamate receptor; nGJ/Cx36, neuronal gap junction coupling and connexin 36; NRSE, neuron-restrictive silencer element; PKA, protein kinase A; PKC, protein kinase C; 3′UTR, 3′-untranslated region; VGCC, voltage-gated Ca²⁺ channels; ⊕, activation of group II mGluRs or GABA_ARs; ⊖, inactivation of group II mGluRs or GABA_ARs; ↑, increase; ↓, decrease. See text for details.

Neuron-restrictive silencer element (NRSE) is a DNA sequence element in a promoter region of a number of neuronal genes (including the Cx36 gene) that binds RE1-silencing transcription factor and regulates the transcriptional activity of these genes.^14,15 Our results with the use of reverse-transcription quantitative real-time polymerase chain reaction and luciferase reporter activity analysis suggested that the receptor-dependent increase in Cx36 expression, that is mediated in developing neurons by activation of group II mGluRs or inactivation of GABA_ARs, is regulated via removal of the NRSE-dependent repression of the Cx36 gene promoter activity. In contrast, the receptor-dependent decrease in Cx36 expression, that is mediated by inactivation of group II mGluRs or activation of GABA_ARs, likely involves post-transcriptional mechanisms dependent upon sequences within the 3′-untranslated region of the Cx36 mRNA (Fig. 1).

In the developing CNS, programmed cell death helps to establish the final number of neurons and contributes to distribution of various cell classes and neuronal circuit formation.¹⁶ The activity of NMDA receptors also is the factor that plays a role in cell survival versus death decisions during neuronal development.^17,18 It has been suggested that during development gap junctions are involved in the regulation of apoptosis¹⁹ and NMDA receptor-dependent neuronal death.⁸ In our study, using methyl thiazolyl tetrazolium assay in developing rat hypothalamic and mouse cortical cultures, we found that prolonged activation of group II mGluRs, that augments the developmental increase in neuronal gap junction coupling, also makes neurons significantly more susceptible to the NMDA receptor-mediated excitotoxicity. At the same time, prolonged inactivation of group II mGluRs, that prevents the developmental increase in neuronal gap junction coupling, also completely prevents the NMDA receptor-mediated neuronal cell death. This suggests that mechanisms for the developmental increase in neuronal gap junction coupling directly regulate death/survival mechanisms in developing neurons.

Altogether, the results revealed a multi-tiered strategy for developing chemical synapses in regulation of electrical synapses. The results also indicated an important role for the mechanisms of regulation of gap junction coupling in the control of death mechanisms in developing neurons.

Abbreviations

CNS

central nervous system

Cx36

connexin 36

GABA_AR

γ-aminobutyric acid A receptor

mGluR

metabotropic glutamate receptor

NMDA

N-methyl-D-aspartate

NRSE

neuron-restrictive silencer element

Addendum to: Park WM, Wang Y, Park S, Denisova JV, Fontes JD, Belousov AB. Interplay of chemical neurotransmitters regulates developmental increase in electrical synapses. J Neurosci. 2011;31:5909–5920. doi: 10.1523/JNEUROSCI.67.