Serotonergic neurons signal reward and punishment on multiple timescales

Serotonin's function in the brain is unclear. One challenge in testing the numerous hypotheses about serotonin's function has been observing the activity of identified serotonergic neurons in animals engaged in behavioral tasks. We recorded the activity of dorsal raphe neurons while mice experienced a task in which rewards and punishments varied across blocks of trials. We ‘tagged’ serotonergic neurons with the light-sensitive protein channelrhodopsin-2 and identified them based on their responses to light. We found three main features of serotonergic neuron activity: (1) a large fraction of serotonergic neurons modulated their tonic firing rates over the course of minutes during reward vs punishment blocks; (2) most were phasically excited by punishments; and (3) a subset was phasically excited by reward-predicting cues. By contrast, dopaminergic neurons did not show firing rate changes across blocks of trials. These results suggest that serotonergic neurons signal information about reward and punishment on multiple timescales

Serotonergic neurons signal reward and punishment on multiple timescales

Serotonin's function in the brain is unclear. One challenge in testing the numerous hypotheses about serotonin's function has been observing the activity of identified serotonergic neurons in animals engaged in behavioral tasks. We recorded the activity of dorsal raphe neurons while mice experienced a task in which rewards and punishments varied across blocks of trials. We ‘tagged’ serotonergic neurons with the light-sensitive protein channelrhodopsin-2 and identified them based on their responses to light. We found three main features of serotonergic neuron activity: (1) a large fraction of serotonergic neurons modulated their tonic firing rates over the course of minutes during reward vs punishment blocks; (2) most were phasically excited by punishments; and (3) a subset was phasically excited by reward-predicting cues. By contrast, dopaminergic neurons did not show firing rate changes across blocks of trials. These results suggest that serotonergic neurons signal information about reward and punishment on multiple timescales. DOI: http://dx.doi.org/10.7554/eLife.06346.00

Maturation of Spinal Motor Neurons Derived from Human Embryonic Stem Cells

Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP) in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases

Spike frequency adaptation (SFA) and rebound action potentials (RAPs) in hESMNs.

<p>(A) An example of the change in instantaneous frequency during a train of action potentials evoked with positive current injection for 1 sec. Inset shows APs (upper) and injected currents (bottom). APs from which ‘a’ and ‘b’ ISI values were measure for SFA calculation are indicated. (B) SFA ratio, calculated as the maximum value of normalized ISIs after any amplitude of positive current injection, increased with DIV (n  = 8, R  = 0.73, P<0.05, Pearson’s linear regression). (C) RAPs were observed in a large subset of hESMNs. Upper trace shows voltage change after negative current injection. Bottom trace shows injected negative current steps. RAP follows the return of current to baseline after the hyperpolarizing step. (D) Incidence of RAPs in hESMNs at 4 different ages as indicated in Fig. 3 legend (n  = 29). Negative current steps with 5 pA increments (to at least 20 pA) were injected while checking for RAPs.</p

Developmental changes in intrinsic membrane properties of hESMNs.

<p>(A) Input resistance decreased over days <i>in vitro</i> (n  = 28, P<0.01, one-way ANOVA). ∗∗ P<0.01, Tukey’s <i>post hoc</i> test. (B) Resting membrane potential and (C) rheobase did not change (n  = 27 and 29, respectively). Positive current steps were injected in 5 pA increments to distinguish small differences in rheobase among individual neurons. (D) Half-width of action potentials (APs),, changed over time <i>in vitro</i> (n  = 26, P<0.001, one-way ANOVA). ∗∗ P<0.01, ∗∗∗ P<0.001, Tukey’s <i>post hoc</i> test. (E) Maximum frequency of APs after current injection increased over time <i>in vitro</i> (n  = 28, P<0.05, one-way ANOVA). ∗P<0.05, Tukey’s <i>post hoc</i> test. Dots shows frequency values for individual neurons. The numbers in parentheses indicate the number of neurons used for analysis taken from 22 dishes in total. In all panels, the first bar represents data from 31+2 DIV, 2^nd bar is 31+4 DIV, 3^rd bar is 31+8/31+9 DIV and 4^th bar is 31+12/31+13 DIV.</p

Representative morphology and membrane potential responses to current step injection in hESMNs at 3 different times <i>in vitro</i>.

<p>Imaging of cells fixed after patch-clamp recordings indicate that recorded cells express the <i>Hb9</i>::GFP reporter transgene (A-C,E,G,and I). Voltage responses and imaging in the same rows are taken from same neurons. The neurons for A-C are same as that shown in F and G. D,F,H show examples of voltage responses to current steps recorded from 3 neurons current-clamped at −58 mV, −60 mV, and −55 mV, respectively. Bottom traces in D,F, and H show injected currents. Scale bars in images are 50 μm.</p

Human ES-derived motor neurons show increasing morphological complexity as they mature <i>in vitro</i>.

<p>(A) Top, schematic of ES cell directed differentiation to motor neurons shows timing of addition of the inductive cues retinoic acid (RA), and sonic hedgehog (SHH). Bottom, timing of morphometric and electrophysiological analyses. (B) Representative image of day 31+5 hESMN showing mature neuronal morphology and co-expression of GFP with motor neuron marker HB9. GFP intensity distinguished hESMN cell bodies (arrow, ∼65,000 gray levels (g.l.)), neurites (arrowhead, ∼18,000 g.l.), and cytoplasmic GFP background in non-MNs (star, ∼800 g.l.). Scale bar 50 µm. (C) Representative camera lucida (Metamorph) neurite traces from 5 randomly chosen (every 8^th) image fields at day 31+2, 31+5, and 31+9 show increasing neurite size and complexity. Scale bar 40 µm. (D-F) Soma area, branches, total neurite outgrowth and processes (not shown) were quantified (number of cells analyzed at each timepoint shown in brackets in D), median (grey line), mean (red line), 25–75 percentile (grey box), 10–90 percentiles (whisker bars), all outliers (+) are shown for each day from which measurements were made. The values for each morphometric parameter on each day were distributed non-normally (Shapiro-Wilk test, P<0.05) and Kruskal-Wallis One Way Analysis of Variance on Ranks showed significant changes in (D) cell soma area (H = 43.885, 2 d.f., P<0.001), (E) complexity or branches/cell H = 309.245, 2 d.f., P<0.001), (F) total neurite outgrowth (H = 161.287, 2 d.f., P<0.001), and (not shown) number of primary neurites (median, 25^th–75^th percentile: day 33: 3, 2–5; day 36: 6, 5–9; day 40: 8, 6–12, H = 442.555, 2 d.f., P<0.001). All significant <i>post hoc</i> pairwise comparisons, Dunn’s Method, are shown by black bars on graphs. and all pairwise comparisons for primary neurite number were significant, P<0.05.</p

Accelerated High-Yield Generation of Limb-Innervating Motor Neurons from Human Stem Cells

Human pluripotent stem cells are a promising source of differentiated cells for developmental studies, cell transplantation, disease modeling, and drug testing. However, their widespread use even for intensely studied cell types like spinal motor neurons is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report a combination of small molecules that within 3 weeks induce motor neurons at up to 50% abundance and with defined subtype identities of relevance to neurodegenerative disease. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1, and column-specific markers that mirror those observed in vivo in human embryonic spinal cord. They also exhibited spontaneous and induced activity, and projected axons toward muscles when grafted into developing chick spinal cord. Strikingly, this novel protocol preferentially generates motor neurons expressing markers of limb-innervating lateral motor column motor neurons (FOXP1+/LHX3−). Access to high-yield cultures of human limb-innervating motor neuron subtypes will facilitate in-depth study of motor neuron subtype-specific properties, disease modeling, and development of large-scale cell-based screening assays

Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines

The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines