External tufted cells drive the output of olfactory bulb glomeruli.

Odors synchronize the activity of olfactory bulb mitral cells that project to the same glomerulus. In vitro, a slow rhythmic excitation intrinsic to the glomerular network persists, even in the absence of afferent input. We show here that a subpopulation of juxtaglomerular cells, external tufted (ET) cells, may trigger this rhythmic activity. We used paired whole-cell recording and Ca(2+) imaging in bulb slices from wild-type and transgenic mice expressing the fluorescent Ca(2+) indicator protein GCaMP-2. Slow, periodic population bursts in mitral cells were synchronized with spontaneous discharges in ET cells. Moreover, activation of a single ET cell was sufficient to evoke population bursts in mitral cells within the same glomerulus. Stimulation of the olfactory nerve induced similar population bursts and activated ET cells at a lower threshold than mitral cells, suggesting that ET cells mediate feedforward excitation of mitral cells. We propose that ET cells act as essential drivers of glomerular output to the olfactory cortex.journal articleresearch support, non-u.s. gov't2009 Feb 18importe

Connexin36 mediates spike synchrony in olfactory bulb glomeruli

SummaryNeuronal synchrony is important to network behavior in many brain regions. In the olfactory bulb, principal neurons (mitral cells) project apical dendrites to a common glomerulus where they receive a common input. Synchronized activity within a glomerulus depends on chemical transmission but mitral cells are also electrically coupled. We examined the role of connexin-mediated gap junctions in mitral cell coordinated activity. Electrical coupling as well as correlated spiking between mitral cells projecting to the same glomerulus was entirely absent in connexin36 (Cx36) knockout mice. Ultrastructural analysis of glomeruli confirmed that mitral-mitral cell gap junctions on distal apical dendrites contain Cx36. Coupled AMPA responses between mitral cell pairs were absent in the knockout, demonstrating that electrical coupling, not transmitter spillover, is responsible for synchronization. Our results indicate that Cx36-mediated gap junctions between mitral cells orchestrate rapid coordinated signaling via a novel form of electrochemical transmission

miR-132 mediates the integration of newborn neurons into the adult dentate gyrus.

Neuronal activity enhances the elaboration of newborn neurons as they integrate into the synaptic circuitry of the adult brain. The role microRNAs play in the transduction of neuronal activity into growth and synapse formation is largely unknown. MicroRNAs can influence the expression of hundreds of genes and thus could regulate gene assemblies during processes like activity-dependent integration. Here, we developed viral-based methods for the in vivo detection and manipulation of the activity-dependent microRNA, miR-132, in the mouse hippocampus. We find, using lentiviral and retroviral reporters of miR-132 activity, that miR-132 is expressed at the right place and right time to influence the integration of newborn neurons. Retroviral knockdown of miR-132 using a specific 'sponge' containing multiple target sequences impaired the integration of newborn neurons into the excitatory synaptic circuitry of the adult brain. To assess potential miR-132 targets, we used a whole-genome microarray in PC12 cells, which have been used as a model of neuronal differentiation. miR-132 knockdown in PC12 cells resulted in the increased expression of hundreds of genes. Functional grouping indicated that genes involved in inflammatory/immune signaling were the most enriched class of genes induced by miR-132 knockdown. The correlation of miR-132 knockdown to increased proinflammatory molecular expression may indicate a mechanistic link whereby miR-132 functions as an endogenous mediator of activity-dependent integration in vivo

miR-132 Sponge Efficacy and Specificity.

<p>To test the efficacy of the sponge we examined whether it altered miR-132 expression in rat PC12 cells. <b>a,</b> Treatment of PC12 cells with NGF resulted in a 1.7 fold increase in mature miR-132 expression (p<.001, Newman-Keul test). Infection with the miR-132 sponge prevented this miR-132 induction. To determine the effect of miR-132 induction on a target, we measured mCherry expression in a monoclonal PC12 cell-line expressing the miR-132 inverse reporter. As expected, NGF treatment significantly reduced mCherry expression and this reduction was blocked by the miR-132 sponge. <b>b,</b> To test the specificity of the sponge we examined the microRNA expression profile of HEK293 cells infected with the miR-132 sponge. We selected HEK293 cells because they have a very low abundance of miR-132, thus limiting possible indirect effects on other microRNAs as a result of miR-132 knockdown. Using the TaqMan multiplex array we robustly detected 86 microRNAs (Ct<34). Of these, only seven were reduced by more than 25% in the sponge-expressing cells. We cross-referenced these seven microRNAs to microarray data examining microRNAs expressed in the adult dentate gyrus (Luikart, unpublished data). Only three microRNAs were both reduced in the multiplex array and expressed endogenously in the dentate gyrus – miR-187, miR-218, and miR-301. A single multiplex array can yield highly variable results. We therefore assessed whether these potential off-target effects could be validated using larger sample sizes (n = 3 independent experiments) and real-time PCR. <b>c,</b> Infection of PC12 cells with the miR-132 sponge resulted in a 32% reduction in miR-132 expression, but did not reduce the expression of miRs-187, 218, 301, or 212 (p>0.3; ANOVA with Tukey post-hoc). miR-212 is highly homologous to miR-132 and generated from the same transcript, making it a particularly good specificity control.</p

miR-132 Expression Correlated with Maturation of Newborn Granule Neurons in the Adult Dentate Gyrus.

<p>An equal titer of lentiviral inverse reporter for miR-132 or the reporter control was injected into the 6 to 8-week-old mouse dentate gyrus and the expression pattern compared at 7 days post-injection. <b>a,b,</b> The miR-132 inverse reporter and the inverse reporter control (red) were both highly expressed in nestin immunopositive progenitor cells (green). <b>c,d,</b> In the subset of progenitor cells and newborn neurons labeled by doublecortin immunostaining (green), there were fewer doublecortin positive cells expressing the miR-132 inverse reporter than the control. <b>e,f,</b> In NeuN immunopositive mature granule cells (green), there was virtually no overlap with expression of the miR-132 inverse reporter (red), whereas the control was expressed in numerous mature granule cells. <b>g,</b> The analysis of co-labeled cells showed significant decreases in expression of the inverse reporter in doublecortin (DCX)- and NeuN labeled cells. The data are expressed as a percentage of the immunolabeled-labeled cells that also expressed the inverse reporter or reporter control. A smaller percentage of NeuN than nestin or DCX positive cells were labeled with the control virus, presumably because of the much larger number of mature granule cells. <b>h,</b> Using fluorescence activated cell sorting of nestin-GFP or POMC-GFP transgenic animals, we isolated purified populations of precursor cells (black bars; nestin-GFP cells), newborn neurons (gray bars; POMC-GFP cells), and the cell fraction containing mature neurons (white bars; unlabeled cells from POMC-GFP animals). The relative transcript levels of pri-miR-132, nestin, and calbindin were measured using real-time PCR. The miR-132 transcript was highest in mature neurons as supported by the expression of calbindin transcript, whereas the nestin transcript was highest in precursor cells.</p

miR-132 Knockdown Correlates to Increased Expression of Molecules Regulated by Immune Signaling Pathways.

<p>miR-132 was knocked down in PC12 cells infected with the miR-132 sponge virus (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019077#pone-0019077-g004" target="_blank">Figure 4</a>). Whole rat genome microarrays were probed to screen for changes in miR-132 knockdown cells vs. control-infected cells. <b>a,</b> Functional grouping of genes that increased when miR-132 was knocked down indicated a significant enrichment in genes regulated by signaling pathways that are involved in immune function. The pie chart represents all categories for the upregulated genes. The labeled categories are the 8 most significantly enriched. <b>b,</b> Using real-time PCR we confirmed an increase in 4 out of 5 genes that were upregulated on the microarray. <b>c,</b> At 21 days post-injection miR-132 sponge expressing neurons (green) had an increase in IL-6 immunofluorescence (compare blue in white outlined areas in 3rd panel from the left) as compared to control redRubi infected neurons (red), consistent with the result for IL-6 in the microarray.</p

An Inverse Reporter for Detecting miR-132 Expression <i>In Vivo</i>.

<p><b>a,</b> We generated an inverse miR-132 reporter lentivirus by placing two perfectly complementary miR-132 target sequences (miR-132 MRE) downstream of mCherry driven by an internal ubiquitin promoter (pUbiquitin). This inverse reporter design results in suppression of mCherry expression in the presence of miR-132. <b>b,</b> The reporter control virus was generated by placing the reverse complement of the miR-132 target (RC miR-132 MRE) downstream of mCherry. <b>c</b>, Hek293 cells were infected with either the inverse reporter or reporter control lentivirus. Single cells were cultivated to generate clonal cell lines expressing the miR-132 inverse reporter (top row) or the inverse reporter control (bottom row). Infection of a proportion of the cells with a miR-132-expressing retrovirus (green) suppressed mCherry in the inverse reporter cells (black silhouettes in red panel, top row), but not in inverse reporter control cells (red panel, bottom row). <b>d,</b> We co-injected equal titers of the GFP-expressing FUGW virus and either the mCherry-expressing miR-132 inverse reporter virus or the reporter control virus into 8 week old mice. FUGW lentivirus showed widespread infection of granule cells at 7 days post-injection (DPI, left panels). In contrast, the mCherry expressing miR-132 inverse reporter virus primarily labeled cells along the subgranular zone of the dentate gyrus (top row, middle panel). The merge is shown in the right panel. Infection of the reporter control virus showed the same widespread infection as the GFP-expressing FUGW virus (lower row, left and middle panel).</p

In Paired Recording, Evoked AMPA Currents Were Smaller in miR-132 Knockdown Granule Cells.

<p><b>a,</b> We injected equal titers of the mCherry expressing control retrovirus (redRubi) and the EGFP expressing miR-132 sponge retrovirus (miR-132sp) <i>in vivo</i>, which resulted in the labeling of control granule cells (red) situated in close proximity to miR-132 knockdown cells (green), as shown for a brain slice on the electrophysiology set-up. <b>b,</b> To activate common inputs to both cells, we placed a stimulating electrode in the perforant path and made simultaneous recordings from control (red) and sponge-expressing cells (green). <b>c,d,</b> The evoked EPSC was much smaller in granule cells expressing the sponge (green) compared to control (red). <b>e,f,</b> The paired pulse ratio (PPR) did not differ between control and sponge (green) cells (p = .82 , paired t-test, n = 4 pairs). All recordings were at −70 mV in solution containing 1 mM Mg²⁺ and SR-95531.</p

miR-132 Knockdown Decreases Dendritic Spine Density <i>In Vivo</i>.

<p><b>a,</b> We assessed the effects of miR-132 knockdown on the dendrites of newborn neurons using a retroviral miR-132 sponge (miR-132sp) with four perfect miR-132 targets downstream of the U6 promoter and with EGFP downstream of the ubiquitin promoter. <b>b,</b> At 21 days post-injection, dendritic morphology was not altered in cells expressing miR-132sp as compared to cells expressing EGFP alone (pRubi, see text for details). <b>c,</b> We compared dendritic spine density in segments from the outer molecular layer in control (pRubi) expressing cells compared to cells expressing the sponge. <b>d,</b> Dendritic spine density decreased in cells expressing the sponge (green) compared to the control (black) (dendritic segments from n = 3 mice, control; 24 dendritic segments from n = 4 mice, miR-132sp; p = .004, t test). Analysis of the variances among dendritic segments showed that there was no difference between animals for control or sponge mice (Levene's test, p = .42).</p