Functional Convergence of Neurons Generated in the Developing and Adult Hippocampus

The dentate gyrus of the hippocampus contains neural progenitor cells (NPCs) that generate neurons throughout life. Developing neurons of the adult hippocampus have been described in depth. However, little is known about their functional properties as they become fully mature dentate granule cells (DGCs). To compare mature DGCs generated during development and adulthood, NPCs were labeled at both time points using retroviruses expressing different fluorescent proteins. Sequential electrophysiological recordings from neighboring neurons of different ages were carried out to quantitatively study their major synaptic inputs: excitatory projections from the entorhinal cortex and inhibitory afferents from local interneurons. Our results show that DGCs generated in the developing and adult hippocampus display a remarkably similar afferent connectivity with regard to both glutamate and GABA, the major neurotransmitters. We also demonstrate that adult-born neurons can fire action potentials in response to an excitatory drive, exhibiting a firing behavior comparable to that of neurons generated during development. We propose that neurons born in the developing and adult hippocampus constitute a functionally homogeneous neuronal population. These observations are critical to understanding the role of adult neurogenesis in hippocampal function

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Unique potential of immature adult-born neurons for the remodeling of CA3 spatial maps

Summary: Mammalian hippocampal circuits undergo extensive remodeling through adult neurogenesis. While this process has been widely studied, the specific contribution of adult-born granule cells (aGCs) to spatial operations in the hippocampus remains unknown. Here, we show that optogenetic activation of 4-week-old (young) aGCs in free-foraging mice produces a non-reversible reconfiguration of spatial maps in proximal CA3 while rarely evoking neural activity. Stimulation of the same neuronal cohort on subsequent days recruits CA3 neurons with increased efficacy but fails to induce further remapping. In contrast, stimulation of 8-week-old (mature) aGCs can reliably activate CA3 cells but produces no alterations in spatial maps. Our results reveal a unique role of young aGCs in remodeling CA3 representations, a potential that can be depleted and is lost with maturation. This ability could contribute to generate orthogonalized downstream codes supporting pattern separation

Slow-Dendritic sIPSCs

<div><p>(A) and (B) Example of traces of outward sIPSCs recorded from a pup (A) and adult (B) DGC. Dashed box on each top trace denotes expanded segment on the bottom. Scales indicate 0.5 s/50 ms (top/bottom), 10 pA.</p> <p>(C) and (D) Two-dimensional histograms of rise and decay time of individual sIPSCs recorded from pup ([C] <i>n</i> = 695 events) and adult DGCs ([D] <i>n</i> = 1,160 events). Color scale indicates the relative frequency for each bin.</p> <p>(E) Cumulative histograms of rise and decay time of all sIPSCs recorded from pup (green) and adult DGCs (red). Same data as shown in (C) and (D).</p> <p>(F) Frequency of sIPSCs (pup, <i>n</i> = 10 neurons; adult, <i>n</i> = 16; <i>p</i> = 0.94; <i>t</i>-test).</p> <p>(G) Peak amplitude of sIPSCs (pup, <i>n</i> = 10; adult, <i>n</i> = 14; <i>p</i> = 0.44).</p> <p>(H) Kinetics of sIPSCs. Inset: scaled averages of sIPSCs (pup, green; adult, red). Scale bar indicates 50 ms. All experiments conducted in the presence of kyn at V_hold = 0 mV with an internal solution containing high [Cl⁻]. (<i>n</i>, same as in [F]; rise time, <i>p</i> = 0.37; decay time, <i>p</i> = 0.41).</p></div

Short-Term Plasticity of Entorhinal Glutamatergic Afferents

<div><p>(A) Average EPSCs recorded at V_Hold = −80 mV (downward deflections) and +50 mV (upward deflections) from pup and adult DGCs (<i>n</i> = 8 to 11) upon stimulation of MPP or LPP. Dashed line indicates zero level. Arrowheads denote time points for quantification of AMPA (open triangles) and NMDA (filled triangles) currents shown in (B). Criteria for AMPA/NMDA quantification are detailed in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040409#s4" target="_blank">Materials and Methods</a> section. Scale bars indicate 20 ms, 100 pA.</p> <p>(B) AMPA/NMDA ratio from pup and adult DGCs (<i>n</i> = 9 to 13) in response to MPP or LPP stimulation. Two-way ANOVA revealed a significant effect of MPP versus LPP (<i>p</i> = 0.006), but no significant effect of pup versus adult (<i>p</i> = 0.63).</p> <p>(C) Averages of EPSCs in response to paired-pulse stimulation of the MPP or LPP delivered at increasing interpulse intervals (20, 50, 100, and 500 ms). Traces are averages of 7–14 cells aligned and normalized to the first EPSC. Stimulation artifacts and late decay phases of the second EPSC were removed for clarity. Scale bar indicates 100 ms.</p> <p>(D) Paired-pulse ratio as a function of interpulse interval for the experiments shown in (C). Two-way ANOVAs revealed a significant effect of interpulse interval for MPP (dashed lines, <i>p</i> < 0.0001) and LPP (solid lines, <i>p</i> < 0.0001), but no significant effect of pup (green lines, solid circles) versus adult (red lines, open circles) for either MPP (<i>p</i> = 0.073) or LPP (<i>p</i> = 0.72) stimulation (<i>n</i> = 9 to 14)</p> <p>(E) Example of EPSCs from a pup (green) and an adult DGC (red) in response to MPP stimulation (ten pulses, 50 Hz) Traces are normalized to the first EPSC amplitude. Scale bar indicates 40 ms.</p> <p>(F) Relative EPSC amplitudes measured in response to 50-Hz stimulation evoked as shown in (E). No difference was found between pup and adult responses (two-way ANOVA, <i>p</i> = 0.49, <i>n</i> = 10 pups [solid circles], <i>n</i> = 4 adults [open circles]). All recordings were carried out in the presence of 20-μm BMI. Neurons were approximately 18 wk old (pup) and approximately 13 wk old (adult). All plots depict mean ± SEM.</p></div

Fluorescent Labeling of DGCs Born during Early Postnatal and Adult Neurogenesis

<div><p>(A) and (B) Retroviral delivery of GFP into DGCs generated at P7 (A) and P42 (B), analyzed 7 wk after each injection. The GCL was labeled by immunohistochemistry for the neuronal marker NeuN (blue). Images are merges of 27 (A) and 21 (B) confocal planes taken from coronal sections (40-μm thick). H, hilus; ML, molecular layer.</p> <p>(C) and (D) Double retroviral labeling of DGCs generated at P7 (GFP⁺, green) and P42 (RFP⁺, red). Images are merges of nine (C) and 20 (D) confocal planes taken from fixed transverse sections of the DG (400-μm thick) from 13-wk-old mice.</p> <p>(E) Double labeling of DGCs with GFP (green) and BrdU (red): intrahippocampal injections of CAG-GFP retrovirus in P7 were followed by daily injections of BrdU carried out from P21 to P25; brains were analyzed at P53. The image is a merge of 16 confocal planes.</p> <p>(F) Example of a sporadic event of co-localization of GFP, BrdU, and NeuN shown by a single optical section for the green, red, and blue channels. Their overlay is shown together with the orthogonal projections onto the <i>x-z</i> (top) and <i>y-z</i> (right) planes.</p> <p>(G) Number of GFP⁺ or BrdU⁺ cells per mouse (left) and the percentage of GFP⁺ cells showing BrdU label (right). Data are mean ± standard error of the mean (SEM) (<i>n</i> = 3 mice). Scale bars indicate 50 μm (A–E) or 10 μm (F).</p></div

Firing Behavior Elicited by Excitatory Inputs

<div><p>(A) Action currents in cell-attached configuration recorded from an adult-born DGC in response to MPP stimulation at increasing stimulus strengths (0.5–1.5 mA, 50 μs). Six representative epochs are shown. Spiking probability (p) is shown below the traces. The asterisk (*) marks the stimulation artifact. Scale indicates 10 ms, 50 pA.</p> <p>(B) Sample experiment of simultaneous cell-attached recordings of DGCs born in pup and adult brain in response to MPP stimulation (0.4 mA, 50 μs). Action currents indicate a higher spiking probability in the pup DGC. Scales indicate 10 ms, 50 pA (left) and 20 pA (right).</p> <p>(C) Sample experiment in which the spiking probability is higher in the adult-born DGC (1.5 mA, 50 μs). Scale indicates 10 ms, 30 pA.</p> <p>(D) Firing behavior of DGCs born in pup and adult brain during simultaneous paired experiments. No significant difference was found (<i>n</i> = 14 pairs, <i>p</i> = 0.8, Wilcoxon signed rank test). All recordings were carried out in the presence of BMI (20 μM). In this set of experiments, adult-born neurons were retrovirally labeled with GFP, whereas unlabeled DGCs of the middle third of the GCL were considered postnatally born (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040409#s4" target="_blank">Materials and Methods</a>). Repetitive (>15 episodes) slow frequency stimulation was used to measure the spiking probability for each neuron at the given stimulus.</p></div

Entorhinal Glutamatergic Afferents onto Mature Neurons Generated in the Developing and Adult Hippocampus

<div><p>(A) Schematic diagram of experimental design for retroviral labeling and electrophysiological recordings.</p> <p>(B) A paired experiment. Upper row: a RFP⁺ neuron was patched (cell “1”) and filled with Alexa Fluor 488 (green). Lower row: a neighboring GFP⁺ neuron (cell “2”) was subsequently patched and filled with Alexa Fluor 594 (red). Both DGCs are in the same field, although at different focal planes (see merge channel). At the end of the experiment, both cells display green and red fluorescence (merge). Scale bar indicates 10 μm.</p> <p>(C) Schematic diagram of the DG showing the position of the bipolar electrodes for the stimulation of the MPP and LPP.</p> <p>(D) Example of EPSCs from an adult-born DGC at V_hold = +50 mV (top) and −80 mV (bottom) elicited by LPP stimulation recorded in the presence of vehicle (“control”) or 20 μM DNQX + 100 μM AP-5. Scale bars indicate 20 ms, 40 pA (top) or 20 pA (bottom). Similar properties were observed in DGCs born during development (unpublished data)</p> <p>(E) Example of a paired experiment. Sequential recordings of two neighboring pup and adult DGCs in response to paired-pulse stimulation (100-ms interval) of the MPP and LPP. Scale bars represent 25 ms, 50 pA.</p> <p>(F) Paired-pulse ratio (left) and 20%–80% rise time (right) of EPSCs recorded from pup and adult DGCs upon stimulation of MPP and LPP (<i>n</i> = 11 to 15) Paired-pulse ratio was measured as the ratio between the amplitude of the second pulse over the first. Two-way analysis of variance (ANOVA) revealed a significant effect of MPP versus LPP for paired-pulse ratio (<i>p</i> = 0.0002) and rise time (<i>p</i> = 0.0017), but no significant effect of pup versus adult (Adu) for either parameter.</p> <p>(G) Peak EPSC amplitude recorded in paired experiments from DGCs born during development (Pup) and adulthood (Adult) upon stimulation of the MPP (<i>n</i> = 15 pairs, <i>p</i> = 0.3, paired <i>t</i>-test) or LPP (<i>n</i> = 11 pairs, <i>p</i> = 0.049). Mean ± SEM is shown on the sides. Recordings were carried out in the presence of 20 μM BMI in slices obtained from mice aged 19–21 wk. Neurons were approximately 18 wk old (P7) and approximately 13 wk old (P42). All plots depict mean ± SEM.</p></div

Fast-Perisomatic sIPSCs

<div><p>(A) and (B) Example of traces of inward sIPSCs recorded from a pup (A) and adult (B) DGC. Dashed box on each top trace denotes expanded segment on the bottom. Scale bars indicate 1 s/50 ms (top/bottom), 40 pA.</p> <p>(C) and (D) Two-dimensional histograms of rise and decay time of individual sIPSCs recorded from pup ([C] <i>n</i> = 5,871 events) and adult DGCs ([D] <i>n</i> = 8,183 events). Color scale indicates the relative frequency for each bin (square areas in the graph).</p> <p>(E) Cumulative histograms of rise and decay time of all sIPSCs recorded from pup (green) and adult (red) DGCs. Data are the same as shown in (C) and (D).</p> <p>(F) Frequency of sIPSCs (pup, <i>n</i> = 12 neurons; adult, <i>n</i> = 15; <i>p</i> = 0.99; <i>t</i>-test).</p> <p>(G) Peak amplitude of sIPSCs (<i>n</i>, same as in [F]; <i>p</i> = 0.44).</p> <p>(H) Kinetics of sIPSCs. Inset: scaled averages of sIPSCs (pup, green; adult, red). Scale bar indicates 10 ms. All experiments conducted in the presence of kyn at V_hold = −80 mV with an internal solution containing high [Cl⁻]. (<i>n</i>, same as in [F]; rise time, <i>p</i> = 0.96; decay time, <i>p</i> = 0.72).</p></div