Optogenetic photochemical control of designer K+ channels in mammalian neurons.

Currently available optogenetic tools, including microbial light-activated ion channels and transporters, are transforming systems neuroscience by enabling precise remote control of neuronal firing, but they tell us little about the role of indigenous ion channels in controlling neuronal function. Here, we employ a chemical-genetic strategy to engineer light sensitivity into several mammalian K(+) channels that have different gating and modulation properties. These channels provide the means for photoregulating diverse electrophysiological functions. Photosensitivity is conferred on a channel by a tethered ligand photoswitch that contains a cysteine-reactive maleimide (M), a photoisomerizable azobenzene (A), and a quaternary ammonium (Q), a K(+) channel pore blocker. Using mutagenesis, we identify the optimal extracellular cysteine attachment site where MAQ conjugation results in pore blockade when the azobenzene moiety is in the trans but not cis configuration. With this strategy, we have conferred photosensitivity on channels containing Kv1.3 subunits (which control axonal action potential repolarization), Kv3.1 subunits (which contribute to rapid-firing properties of brain neurons), Kv7.2 subunits (which underlie "M-current"), and SK2 subunits (which are Ca(2+)-activated K(+) channels that contribute to synaptic responses). These light-regulated channels may be overexpressed in genetically targeted neurons or substituted for native channels with gene knockin technology to enable precise optopharmacological manipulation of channel function.

Episodic Prenatal Exposure To Ethanol Affects Postnatal Neurogenesis In The Macaque Dentate Gyrus And Visual Recognition Memory.

Fetal alcohol syndrome (FAS) is a prime cause of cognitive dysfunction. The present study tested the hypotheses (a) that gestational ethanol exposure results in deficits in hippocampal-related behaviors and associated neurogenesis and (b) that the period of gastrulation is a time of vulnerability. Pregnant macaques were intubated with ethanol or saline once per week for 3, 6, or 24 weeks. Exposures included or omitted the period of gastrulation. Offspring were given behavioral tests including a Visual-Paired Comparison (VPC), a hippocampal-associated memory task, and euthanized as adolescents. Their dentate gyri were processed for immunohistochemical identification of cells passing through the cell cycle (Ki-67 and proliferating cell nuclear antigen), exiting the cell cycle (p21), or passing through early stages of neuronal morphogenesis (Tuj1). Performance in neurobehavioral tasks was unaffected by ethanol exposure, the notable exception being performance in the VPC that was poorer for macaques exposed to ethanol including gastrulation. Anatomical studies show that the expression of Ki-67 was greater and ratio of p21-positive cells to the ratio of Ki-67-expressing cells was lower in animals in which the ethanol exposure included gastrulation. On the other hand, no ethanol-induced differences in TuJ1 expression were detected. Thus, the dentate gyrus is a bellwether of long-term consequences of gestational ethanol exposure. Targeted effects of ethanol on early neural generation (cell cycle and cycle exit) correlate with the timing-dependent degradation in VPC performance and exposure during gastrulation results in notable deficits. These changes evidence a pattern of fetal programming underlying FAS.