Hippocampal astrocytic neogenin regulating glutamate uptake, a critical pathway for preventing epileptic response.

Epilepsy, a common neurological disorder, is featured with recurrent seizures. Its underlying pathological mechanisms remain elusive. Here, we provide evidence for loss of neogenin (NEO1), a coreceptor for multiple ligands, including netrins and bone morphological proteins, in the development of epilepsy. NEO1 is reduced in hippocampi from patients with epilepsy based on transcriptome and proteomic analyses. Neo1 knocking out (KO) in mouse brains displays elevated epileptiform spikes and seizure susceptibility. These phenotypes were undetectable in mice, with selectively depleted NEO1 in excitatory (NeuroD6-Cre+) or inhibitory (parvalbumin+) neurons, but present in mice with specific hippocampal astrocytic Neo1 KO. Additionally, neurons in hippocampal dentate gyrus, a vulnerable region in epilepsy, in mice with astrocyte-specific Neo1 KO show reductions in inhibitory synaptic vesicles and the frequency of miniature inhibitory postsynaptic current(mIPSC), but increase of the duration of miniature excitatory postsynaptic current and tonic NMDA receptor currents, suggesting impairments in both GABAergic transmission and extracellular glutamate clearance. Further proteomic and cell biological analyses of cell-surface proteins identified GLAST, a glutamate-aspartate transporter that is marked reduced in Neo1 KO astrocytes and the hippocampus. NEO1 interacts with GLAST and promotes GLAST surface distribution in astrocytes. Expressing NEO1 or GLAST in Neo1 KO astrocytes in the hippocampus abolishes the epileptic phenotype. Taken together, these results uncover an unrecognized pathway of NEO1-GLAST in hippocampal GFAP+ astrocytes, which is critical for GLAST surface distribution and function, and GABAergic transmission, unveiling NEO1 as a valuable therapeutic target to protect the brain from epilepsy.

Selective activation of cholinergic basal forebrain neurons induces immediate sleep-wake transitions.

The basal forebrain (BF) plays a crucial role in cortical activation [1, 2]. However, the exact role of cholinergic BF (ch-BF) neurons in the sleep-wake cycle remains unclear [3, 4]. We demonstrated that photostimulation of ch-BF neurons genetically targeted with channelrhodopsin 2 (ChR2) was sufficient to induce an immediate transition to waking or rapid eye movement (REM) sleep from slow-wave sleep (SWS). Light stimulation was most likely to induce behavioral arousal during SWS, but not during REM sleep, a result in contrast to the previously reported photostimulation of noradrenergic or hypocretin neurons that induces wake transitions from both SWS and REM sleep. Furthermore, the ratio of light-induced transitions from SWS to wakefulness or to REM sleep did not significantly differ from that of natural transitions, suggesting that activation of ch-BF neurons facilitates the transition from SWS but does not change the direction of the transition. Excitation of ch-BF neurons during wakefulness or REM sleep sustained the cortical activation. Stimulation of these neurons for 1 hr induced a delayed increase in the duration of wakefulness in the subsequent inactive period. Our results suggest that activation of ch-BF neurons alone is sufficient to suppress SWS and promote wakefulness and REM sleep.

Simplified protocol for isolation of multipotential NG2 cells from postnatal mouse.

BACKGROUND: The functions of NG2 cells have attracted much attention since they were identified. At present, our understanding of their properties and functions is still limited due to the lack of an easy protocol for isolating them from mice. NEW METHOD: In the present study, in postnatal mouse cortical tissue cultures, cell confluence was achieved at DIV 6-8 by frequently changing the medium in the absence of viable neurons, and abundant NG2 cells grew on top of the astrocyte layer before microglia started to thrive. Thus, we developed a simple protocol to separate mouse NG2 cells by shaking the cultures on an orbital shaker at 37 °C for only 3-4h. RESULTS: The yield and purity of NG2 cells were sufficiently high, and the cells displayed immunological and electrophysiological phenotypes typical of NG2 cells. They expressed a large delayed-rectifier K+ current (ID) and a transient A-type K+ current (IA) that were electrophysiologically different from astrocytes and neurons. They showed significantly enhanced chemo-attractive migration after application of GABA. They also showed properties of multipotential neuronal precursor cells and were capable of generating oligodendrocytes (54.2±8.1%), neurons (up to 13.3±6.8%), and astrocytes (93.9±4.3%) under defined conditions. Comparison with Existing Method(s): When compared to other methods available for the isolation of mouse NG2 cells, the procedure we present is simple, relatively fast, and economical. CONCLUSIONS: Overall, we present evidence that this new method for isolating NG2 cells from postnatal mice is simple, economical, and effective.