The dorsal raphe (DR) constitutes a major serotonergic input to the forebrain and modulates diverse functions and brain states, including mood, anxiety, and sensory and motor functions. Most functional studies to date have treated DR serotonin neurons as a single population. Using viral-genetic methods, we found that subcortical- and cortical-projecting serotonin neurons have distinct cell-body distributions within the DR and differentially co-express a vesicular glutamate transporter. Further, amygdala- and frontal-cortex-projecting DR serotonin neurons have largely complementary whole-brain collateralization patterns, receive biased inputs from presynaptic partners, and exhibit opposite responses to aversive stimuli. Gain- and loss-of-function experiments suggest that amygdala-projecting DR serotonin neurons promote anxiety-like behavior, whereas frontal-cortex-projecting neurons promote active coping in the face of challenge. These results provide compelling evidence that the DR serotonin system contains parallel sub-systems that differ in input and output connectivity, physiological response properties, and behavioral functions.

Neural stem cell (NSC) therapy represents a potentially powerful approach for gene transfer in the diseased central nervous system. However, transplanted primary, embryonic stem cell- and induced pluripotent stem cell-derived NSCs generate largely undifferentiated progeny. Understanding how physiologically immature cells influence host activity is critical to evaluating the therapeutic utility of NSCs. Earlier inquiries were limited to single-cell recordings and did not address the emergent properties of neuronal ensembles. To interrogate cortical networks post-transplant, we used voltage sensitive dye imaging in mouse neocortical brain slices, which permits high temporal resolution analysis of neural activity. Although moderate NSC engraftment largely preserved host physiology, subtle defects in the activation properties of synaptic inputs were induced. High-density engraftment severely dampened cortical excitability, markedly reducing the amplitude, spatial extent, and velocity of propagating synaptic potentials in layers 2-6. These global effects may be mediated by specific disruptions in excitatory network structure in deep layers. We propose that depletion of endogenous cells in engrafted neocortex contributes to circuit alterations. Our data provide the first evidence that nonintegrating cells cause differential host impairment as a function of engrafted load. Moreover, they emphasize the necessity for efficient differentiation methods and proper controls for engraftment effects that interfere with the benefits of NSC therapy.

Human neural stem cell (hNSC) transplantation improves recovery in preclinical stroke models. However their effects on surviving sensorimotor circuits are not well understood. Here we performed a comprehensive electrophysiological assessment and RNA-seq analysis of the stroke-injured rat cortex after transplantation of two hNSC lines, G010 (fetal-derived) and NR1 (hES-derived). Vehicle, G010, or NR1 cells were transplanted into the ischemic cortex of Nude rats 1 wk after distal middle cerebral artery occlusion. Neurological recovery was accessed by the Whisker-paw test. Acute brain slices were prepared 1 wk post-transplantation for electrophysiological recording. A linear multichannel recording probe was placed in the peri-infarct motor cortex and local field potentials (LFPs) recorded simultaneously from all cortical layers following circuit activation in layer 2/3. For RNA-seq analysis, the transplantation area was dissected, RNA extracted and cDNA libraries prepared for RNA-seq. G010 and NR1 cells enhanced post-stroke behavioral recovery starting 1 or 3 wks post-transplantation, respectively. Current source density (CSD) analysis of evoked LFPs, a method used to more accurately localize synaptic currents, revealed that both G010 and NR1 cells restored circuit excitability in layer 2/3, through reduction in inhibitory/excitatory (I/E) balance. However, the mechanisms driving the I/E balance shift were different, with NR1 cells enhancing excitation while G010 cells reduced inhibition. RNA-seq analysis of the cortex revealed that stem cell- versus vehicle-treated animals had different gene expression patterns in all cortical layers. Moreover, NR1 and G010 cells affected different genes. Layer 2/3-specific genes significantly affected by G010 grafts include Arpp21, Enpp2, Gbe1, Mylk, Pea15, Pkig, Rcn1, Slit3, Uchl1 and Wfs1; while NR1 grafts altered expression of Hapln4, Kcnc1, Ppp1r1b, Scn1a, Slit3 and Slitrk1. GO analysis revealed that both stem cell treatments activated critical canonical pathways e.g. Actin Cytoskeleton Signaling and Synaptic Long Term Depression. Stem cell transplantation modulates host gene expression and this is associated with increased circuit excitability and motor-sensory function.