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

Effects of ageing and neurodegeneration on neuronal plasticity in the hippocampus

A medida que envejece, el cerebro de los mamíferos sufre modificaciones bioquímicas, estructurales y funcionales que disminuyen la plasticidad de sus circuitos. Estos cambios han sido propuestos como responsables del déficit cognitivo y del aumento en la incidencia de las enfermedades neurodegenerativas que se presentan con la edad. El giro dentado del hipocampo cuenta con una forma de plasticidad singular dada por la producción de nuevas neuronas granulares a lo largo de toda la vida. Mediante transducción retroviral con proteínas reporteras y/o transgenes relacionados con la patogénesis de la enfermedad de Alzheimer se logró utilizar a estas neuronas como vehículo para monitorear los efectos del envejecimiento normal y patológico sobre la plasticidad neuronal. Utilizando inmunofluorescencia y microscopía confocal en ratones wild type envejecidos y en transgénicos modelando la enfermedad de Alzheimer pudimos observar que, aunque el número de neuronas generadas disminuye a lo largo de la vida, estas no se ven afectadas en su migración ni en la densidad de los contactos sinápticos aferentes que desarrollan durante el envejecimiento normal o patológico. Por otro lado, combinando registros electrofisiológicos con análisis morfológico encontramos que la expresión autónoma de célula de diferentes variedades de la proteína precursora de amiloide (APP) altera transitoriamente la forma en que estas neuronas se conectan al circuito del giro dentado. Llamativamente, este efecto parecería no estar mediado por el péptido β amiloide, sino por algún dominio contenido en el extremo C-terminal de APP. En conjunto estos resultados estarían indicando que a pesar de la diversidad de los cambios que se presentan en el cerebro durante el envejecimiento y la neurodegeneración, las neuronas granulares que se desarrollan en el giro dentado bajo tales circunstancias pueden incorporarse anatómica y funcionalmente al circuito. Sin embargo, la dinámica con que se da esta integración se ve afectada por la sobreexpresión de APP en forma autónoma de célula. Alteraciones sutiles como esta podrían subyacer a las manifestaciones cognitivas iniciales encontradas en la enfermedad de Alzheimer.Biochemical, structural and functional changes occurs to the mammalian brain as it ages. These modifications are thought to contribute to the decrease in the plasticity of neuronal circuits. This latter situation has been proposed to underlie the cognitive decline and the increase of neurodegenerative disorders found during ageing. A particular form of plasticity is found in the dentate gyrus of the hippocampus where the continuous addition of new granular neurons takes place through all life. Using retroviral transduction of fluorescent proteins and/or transgenes related to Alzheimer’s disease these neurons were experimentally used as probes to study the effects of normal and pathological ageing on neuronal plasticity. Immunofluoresce labeling and confocal microscopy of wild type aged mice and transgenic models of Alzheimer´s disease showed a strong reduction in the number of newly generated neurons during life. However, nor migration neither afferent synaptic connectivity was affected during ageing or neurodegeneration. Electrophysiological recordings and morphological analyses revealed that cell autonomous expression of different forms of amyloid precursor protein (APP) affect the way that these neurons integrate to the circuit of the dentate gyrus. Notably, this effect seems to be mediated by a domain in the C-terminal region of APP rather than by Aβ peptide itself. Altogether these observations should be indicating that despite the diversity in the changes found in the brain during ageing and neurodegeneration, newlyborn neurons are able to functionally and anatomically incorporate to the circuit. However, the timing of this integration was found to be affected by the cell autonomous overexpression of APP. This kind of subtle alterations would be underlying the early cognitive decline found in Alzheimer’s disease.Fil:Morgenstern, Nicolás A.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

A distinctive layering pattern of mouse dentate granule cells is generated by developmental and adult neurogenesis

New neurons are continuously added throughout life to the dentate gyrus of the mammalian hippocampus. During embryonic and early postnatal development, the dentate gyrus is formed in an outside-in layering pattern that may extend through adulthood. In this work, we sought to quantify systematically the relative position of dentate granule cells generated at different ages. We used 5'-bromo-2'-deoxyuridine (BrdU) and retroviral methodologies to birth date cells born in the embryonic, early postnatal, and adult hippocampus and assessed their final position in the adult mouse granule cell layer. We also quantified both developmental and adult-born cohorts of neural progenitor cells that contribute to the pool of adult progenitor cells. Our data confirm that the outside-in layering of the dentate gyrus continues through adulthood and that early-born cells constitute most of the adult dentate gyrus. We also found that substantial numbers of the dividing cells in the adult dentate gyrus were derived from early-dividing cells and retained BrdU, suggesting that a subpopulation of hippocampal progenitors divides infrequently from early development onward.Fil: Mathews, Emily A.. Salk Institute For Biological Studies; Estados Unidos. University Of California At San Diego; Estados UnidosFil: Morgenstern, Nicolás Andrés. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: Piatti, Veronica del Carmen. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: Zhao, Chunmei. Salk Institute for Biological Studies; Estados UnidosFil: Jessberger, Sebastian. Salk Institute for Biological Studies; Estados UnidosFil: Schinder, Alejandro Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Gage, Fred H.. Salk Institute For Biological Studies; Estados Unido

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

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

GABAergic Inputs Evoked by ML Stimulation

<div><p>(A) Example of evoked IPSCs recorded from a pup-born DGC in response to extracellular stimulation of the ML before (control), during (BMI), and after (wash) application of 20 μM BMI. Scale bars indicate 25 ms, 20 pA. Inset: schematic diagram of the DG showing the position of the bipolar electrode for the stimulation of ML interneurons. Experiments were carried out with internal solution containing low [Cl⁻].</p> <p>(B) Kinetics of IPSCs evoked on pup versus adult DGCs (pup, <i>n</i> = 6; adult, <i>n</i> = 14; rise time, <i>p</i> = 0.85; decay time, <i>p</i> = 0.90, paired <i>t</i>-tests). Inset: scaled averages of IPSCs (pup, green; adult, red). Scale bar indicates 50 ms.</p> <p>(C) and (D) Examples of I-V curves of evoked IPSCs recorded from pup (C) or adult (D) DGCs. Top: sample traces of IPSCs recorded at the holding potentials shown on the right (mV). Scale bar indicates 50 ms, 10 pA. Bottom: I-V plots measured at the time indicated by the arrowheads.</p> <p>(E) E_GABA of IPSCs (pup, <i>n</i> = 7; adult, <i>n</i> = 11; <i>p</i> = 0.67, <i>t</i>-test).</p> <p>(F) Slope conductance measured at the IPSC peak (<i>n,</i> same as in [E]; <i>p</i> = 0.23; <i>t</i>-test). All experiments conducted in the presence of kyn (4 mM) in neurons aged 12 wk (P7) and 7 wk (P42).</p></div