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Axon-dendrite polarization is crucial for neural network wiring and information processing in the brain. Polarization begins with the transformation of a single neurite into an axon and its subsequent rapid extension, which requires coordination of cellular energy status to allow for transport of building materials to support axon growth. We found that activation of the energy-sensing adenosine 5′-monophosphate (AMP)–activated protein kinase (AMPK) pathway suppressed axon initiation and neuronal polarization. Phosphorylation of the kinesin light chain of the Kif5 motor protein by AMPK disrupted the association of the motor with phosphatidylinositol 3-kinase (PI3K), preventing PI3K targeting to the axonal tip and inhibiting polarization and axon growth.

During mitotic division in the telencephalic proliferative ventricular zone (VZ), the nuclei of the neural precursors move basally away from the ventricular surface for DNA synthesis, and apically return to the surface for mitotic division; a process known as interkinetic migration or “to-and-fro” nuclear translocation. The cell, which remains attached to the ventricular surface, either continues cycling, or exits the cycle and migrates to the subventricular zone or the developing cortical plate. Although gap junctions/hemichannels are known to modulate DNA synthesis via Ca2+waves, the role of Ca+oscillations and the mechanism of nuclear translocation in the VZ precursors are unclear. Here, we provide evidence that, during apical nuclear migration, VZ precursors display dynamic spontaneous Ca2+transients, which depend on functional gap junctions/hemichannels via ATP release and Ca2+-mobilizing messenger diffusion. Furthermore, we found that blocking gap junctions/hemichannels or short hairpin RNA-mediated knockdown of Cx43 (connexin 43) retards the apically directed interkinetic nuclear migration accompanied with changes in the nuclear length/width ratio. In addition, we demonstrated that blocking functional gap junctions/hemichannels induces phosphorylation of small GTPase cdc42 in the VZ precursors. The basal phase of interkinetic migration is much slower and appears to be mediated passively by mechanical forces after cell division. Our findings indicate that functional interference with gap junctions/hemichannels during embryonic development may lead to abnormal corticogenesis and dysfunction of the cerebral cortex in adult organisms.

Most neurons of the cerebral cortex are generated in the germinal zones near the embryonic cerebral ventricle and migrate radially to the overlying cortical plate. Initially, all dividing cells are attached to the surface of the embryonic ventricle (ventricular zone) until a subset of dividing cells (basal or intermediate neuronal progenitors, INPs), recognized by their immunoreactivity to Tbr2, detach from the ventricular surface and migrate a short distance to establish a secondary proliferative compartment (the subventricular zone). The mechanism that regulates migration of the Tbr2 + INPs from the ventricular to the subventricular zones is unknown. Here, we show that INPs, unlike the postmitotic neurons that tend to lose the ATP response, continue to express the purinergic P2Y1 receptor. Furthermore, blocking ATP signaling by the P2Y1 blockers, MRS2176, suramin, and apyrase, reduces Ca 2+ transients and retards INP migration to the subventricular zone. In addition, genetic knockdown of the P2Y1 receptor by in vivo application of short hairpin RNA selectively impairs the migration of INPs to the subventricular zone. Together, these results suggest that intercellular ATP signaling is essential for the migration of INPs and the proper formation of the subventricular zone. Interference of ATP signaling or abnormal Ca 2+ fluctuations in INPs may play a significant role in variety of genetic or acquired cortical malformations.

ATP induces pain via activation of purinergic receptors in nociceptive sensory nerves. ATP signaling is terminated by ATP hydrolysis mediated by cell surface-localized ecto-nucleotidases. Using enzymatic histochemical staining, we show that ecto-ATPase activity is present in mouse trigeminal nerves. Using immunofluorescence staining, we found that ecto-NTPDase3 is expressed in trigeminal nociceptive neurons and their projections to the brainstem. In addition, ecto-ATPase activity and ecto-NTPDase3 are also detected in the nociceptive outermost layer of the trigeminal subnucleus caudalis. Furthermore, we demonstrate that incubation with anti-NTPDase3 serum reduces extracellular ATP degradation in the nociceptive lamina of both the trigeminal subnucleus caudalis and the spinal cord dorsal horn. These results are consistent with neuronal NTPDase3 activity modulating pain signal transduction and transmission by affecting extracellular ATP hydrolysis within the trigeminal nociceptive pathway. Thus, disruption of trigeminal neuronal NTPDase3 expression and localization to presynaptic terminals during chronic inflammation, local constriction and injury may contribute to the pathogenesis of orofacial neuropathic pain.

In the postnatal subventricular zone (SVZ), local cues or signaling molecules released from neuroblasts limit the proliferation of glial fibrillary acidic protein (GFAP)-expressing progenitors thought to be stem cells. However, signals between SVZ cells have not been identified. We show that depolarization of neuroblasts induces nonsynaptic SNARE-independent GABA(A) receptor currents in GFAP-expressing cells, the time course of which depends on GABA uptake in acute mouse slices. We found that GABA(A) receptors are tonically activated in GFAP-expressing cells, consistent with the presence of spontaneous depolarizations in neuroblasts that are sufficient to induce GABA release. These data demonstrate the existence of nonsynaptic GABAergic signaling between neuroblasts and GFAP-expressing cells. Furthermore, we show that GABA(A) receptor activation in GFAP-expressing cells limits their progression through the cell cycle. Thus, as GFAP-expressing cells generate neuroblasts, GABA released from neuroblasts provides a feedback mechanism to control the proliferation of GFAP-expressing progenitors by activating GABA(A) receptors.

Neuronal progenitors are continuously generated in the postnatal rodent subventricular zone and migrate along the rostral migratory stream to supply interneurons in the olfactory bulb. Nonsynaptic GABAergic signaling affects the postnatal neurogenesis by depolarizing neuronal progenitors, which depends on an elevated intracellular Cl(-) concentration. However, the molecular mechanism responsible for Cl(-) accumulation in these cells still remains elusive. Using confocal Ca(2+) imaging, we found that GABA depolarization-induced Ca(2+) increase was either abolished by bumetanide, a specific inhibitor of the Na(+) -K(+) -2Cl(-) cotransporter, or reduced by partial replacement of extracellular Na(+) with Li(+) , in the HEPES buffer but not in the CO(2)/HCO₃⁻ buffer. GABA depolarization-induced Ca(2+) increase in CO(2)/HCO₃⁻ buffer was abolished by a combination of bumetanide with the anion exchanger inhibitor DIDS or with the carbonic anhydrase inhibitor acetozalimide. Using gramicidin-perforated patch-clamp recording, we further confirmed that bumetanide, together with DIDS or acetozalimide, reduced the intracellular chloride concentration in the neuronal progenitors. In addition, with BrdU labeling, we demonstrated that blocking of the Na(+) -K(+) -2Cl(-) cotransporter, but not anion exchangers, reduced the proliferation of neuronal progenitors. Our results indicate that both the Na(+) -K(+) -2Cl(-) cotransporter and anion exchangers contribute to the elevated intracellular chloride responsible for the depolarizing action of GABA in the postnatal forebrain neuronal progenitors. However, the Na(+) -K(+) -2Cl(-) cotransporter displays an additional effect on neuronal progenitor proliferation.

Elevated levels of [Formula: see text] in the brain impair neuronal function. We studied the effects of [Formula: see text] on postsynaptic inhibition of cultured rat brain neurons using whole cell recording under nominally [Formula: see text] -free conditions. Application of [Formula: see text] shifted the reversal potentials for spontaneous inhibitory postsynaptic currents and currents elicited by dendritic GABA applications in a positive direction because [Cl–]i increased. The positive shift of the reversal potentials of GABA-induced Cl– currents was equal on equimolar elevation of [Formula: see text] or [K+]o, respectively. The [Formula: see text]-induced increase in [Cl–]i was reversed by an inhibitor of cation-anion cotransport, furosemide (0.1 mM), but not by bumetanide (0.01 mM) or by replacement of [Na+]o by Li+. We conclude that neuron-specific K-Cl cotransporter (KCC2) transports [Formula: see text] similar to K+. Despite this fact, the small increase of [Formula: see text] during metabolic encephalopathies will barely elevate [Cl–]i. However, an impairment of neuronal function may result because KCC2 provides a pathway to accumulate [Formula: see text], and thereby, a continuous acid load to neurons.