Choroid plexus epithelial cells (CPECs) secrete cerebrospinal fluid (CSF). They express Na+-K+-ATPase and Na+-K+-2Cl− cotransporter 1 (NKCC1) on their apical membrane, deviating from typical basolateral membrane location in secretory epithelia. Given this peculiarity, the direction of basal net ion fluxes mediated by NKCC1 in CPECs is controversial, and cotransporter function is unclear. Determining the direction of basal NKCC1-mediated fluxes is critical to understanding the function of apical NKCC1. If NKCC1 works in the net efflux mode, it may be directly involved in CSF secretion. Conversely, if NKCC1 works in the net influx mode, it would have an absorptive function, contributing to intracellular Cl− concentration ([Cl−]i) and cell water volume (CWV) maintenance needed for CSF secretion. We resolve this long-standing debate by electron microscopy (EM), live-cell-imaging microscopy (LCIM), and intracellular Na+ and Cl− measurements in single CPECs of NKCC1+/+ and NKCC1−/− mouse. NKCC1-mediated ion and associated water fluxes are tightly linked, thus their direction is inferred by measuring CWV changes. Genetic or pharmacological NKCC1 inactivation produces CPEC shrinkage. EM of NKCC1−/− CPECs in situ shows they are shrunken, forming large dilations of their basolateral extracellular spaces, yet remaining attached by tight junctions. Normarski LCIM shows in vitro CPECs from NKCC1−/− are ~17% smaller than NKCC1+/+. CWV measurements in calcein-loaded CPECs show that bumetanide (10 μM) produces ~16% decrease in CWV in NKCC1+/+ but not in NKCC1−/− CPECs. Our findings suggest that under basal conditions apical NKCC1 is continuously active and works in the net inward flux mode maintaining [Cl−]i and CWV needed for CSF secretion.

The use of transcranial electrical stimulation (TES) as a method to augment neural activity has increased in popularity in the last decade and a half. The specific application of TES to the left prefrontal cortex has been shown to produce broad cognitive effects; however, the neural mechanisms underlying these effects remain unknown. In this work, we evaluated the effect of repetitive TES on cerebral perfusion. Stimulation was applied to the left prefrontal cortex on three consecutive days, and resting cerebral perfusion was quantified before and after stimulation using arterial spin labeling. Perfusion was found to decrease significantly more in a matched sham stimulation group than in a group receiving active stimulation across many areas of the brain. These changes were found to originate in the locus coeruleus and were broadly distributed in the neocortex. The changes in the neocortex may be a direct result of the stimulation or an indirect result via the changes in the noradrenergic system produced from the altered activity of the locus coeruleus. These findings indicate that anodal left prefrontal stimulation alters the activity of the locus coeruleus, and this altered activity may excite the noradrenergic system producing the broad behavioral effects that have been reported.

Transcranial direct current stimulation (tDCS) to the left prefrontal cortex has been shown to produce broad behavioral effects including enhanced learning and vigilance. Still, the neural mechanisms underlying such effects are not fully understood. Furthermore, the neural underpinnings of repeated stimulation remain understudied. In this work, we evaluated the effects of the repetition and intensity of tDCS on cerebral perfusion [cerebral blood flow (CBF)]. A cohort of 47 subjects was randomly assigned to one of the three groups. tDCS of 1- or 2-mA was applied to the left prefrontal cortex on three consecutive days, and resting CBF was quantified before and after stimulation using the arterial spin labeling MRI and then compared with a group that received sham stimulation. A widespread decreased CBF was found in a group receiving sham stimulation across the three post-stimulation measures when compared with baseline. In contrast, only slight decreases were observed in the group receiving 2-mA stimulation in the second and third post-stimulation measurements, but more prominent increased CBF was observed across several brain regions including the locus coeruleus (LC). The LC is an integral region in the production of norepinephrine and the noradrenergic system, and an increased norepinephrine/noradrenergic activity could explain the various behavioral findings from the anodal prefrontal tDCS. A decreased CBF was observed in the 1-mA group across the first two post-stimulation measurements, similar to the sham group. This decreased CBF was apparent in only a few small clusters in the third post-stimulation scan but was accompanied by an increased CBF, indicating that the neural effects of stimulation may persist for at least 24 h and that the repeated stimulation may produce cumulative effects.