Highly conductive carbon nanotube matrix accelerates developmental chloride extrusion in central nervous system neurons by increased expression of chloride transporter KCC2

Exceptional mechanical and electrical properties of carbon nanotubes (CNT) have attracted neuroscientists and neural tissue engineers aiming to develop novel devices that interface with nervous tissues. In the central nervous system (CNS), the perinatal chloride shift represents a dynamic change that forms the basis for physiological actions of γ-aminobutyric acid (GABA) as an inhibitory neurotransmitter, a process of fundamental relevance for normal functioning of the CNS. Low intra-neuronal chloride concentrations are maintained by a chloride-extruding transporter, potassium chloride cotransporter 2 (KCC2). KCC2's increasing developmental expression underlies the chloride shift. In neural injury, repressed KCC2 expression plays a co-contributory role by corrupting inhibitory neurotransmission. Mechanisms of Kcc2 up-regulation are thus pertinent because of their medical relevance, yet they remain elusive. Here, it is shown that primary CNS neurons originating from the cerebral cortex, cultured on highly-conductive few-walled-CNT (fwCNT) have a strikingly accelerated chloride shift caused by increased KCC2 expression. KCC2 upregulation is dependent on neuronal voltage-gated calcium channels (VGCC) and, furthermore, on calcium/calmodulin-dependent kinase II, which is linked to VGCC-mediated calcium-influx. It is also demonstrated that accelerated Kcc2 transcription in brain-slices prepared from genetically-engineered reporter mice, in which Kcc2 promoter drives luciferase, when the cerebral cortex of these mice is exposed to fwCNT-coated devices. Based on these findings, whether fwCNT can enhance neural engineering devices for the benefit of neural injury conditions associated with elevated neuronal intracellular chloride concentration - such as pain, epilepsy, traumatic neural injury and ischemia - can now be addressed. Taken together, our novel insights illustrate how fwCNTs can promote low neuronal chloride in individual neurons and thus inhibitory transmission in neural circuits. Few-walled carbon nanotubes (fwCNTs) with very high electrical conductivity, when used as culture matrix for primary nerve cells from the central nervous system, force the neurons to lower their chloride faster. Mechanistic insights are provided into how fwCNTs exert their powerful impact on nerve cells' chloride homeostasis, which underlies inhibitory transmission in the central nervous system.