17 research outputs found

    Retrograde Tracing with Recombinant Rabies Virus Reveals Correlations Between Projection Targets and Dendritic Architecture in Layer 5 of Mouse Barrel Cortex

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    A recombinant rabies virus was used as a retrograde tracer to allow complete filling of the axonal and dendritic arbors of identified projection neurons in layer 5 of mouse primary somatosensory cortex (S1) in vivo. Previous studies have distinguished three types of layer 5 pyramids in S1: tall-tufted, tall-simple, and short. Layer 5 pyramidal neurons were retrogradely labeled from several known targets: contralateral S1, superior colliculus, and thalamus. The complete dendritic arbors of labeled cells were reconstructed to allow for unambiguous classification of cell type. We confirmed that the tall-tufted pyramids project to the superior colliculus and thalamus and that short layer 5 pyramidal neurons project to contralateral cortex, as previously described. We found that tall-simple pyramidal neurons contribute to corticocortical connections. Axonal reconstructions show that corticocortical projection neurons have a large superficial axonal arborization locally, while the subcortically projecting neurons limit axonal arbors to the deep layers. Furthermore, reconstructions of local axons suggest that tall-simple cell axons have extensive lateral spread while those of the short pyramids are more columnar. These differences were revealed by the ability to completely label dendritic and axonal arbors in vivo and have not been apparent in previous studies using labeling in brain slices

    RORα Coordinates Reciprocal Signaling in Cerebellar Development through Sonic hedgehog and Calcium-Dependent Pathways

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    AbstractThe cerebellum provides an excellent system for understanding how afferent and target neurons coordinate sequential intercellular signals and cell-autonomous genetic programs in development. Mutations in the orphan nuclear receptor RORα block Purkinje cell differentiation with a secondary loss of afferent granule cells. We show that early transcriptional targets of RORα include both mitogenic signals for afferent progenitors and signal transduction genes required to process their subsequent synaptic input. RORα acts through recruitment of gene-specific sets of transcriptional cofactors, including β-catenin, p300, and Tip60, but appears independent of CBP. One target promoter is Sonic hedgehog, and recombinant Sonic hedgehog restores granule precursor proliferation in RORα-deficient cerebellum. Our results suggest a link between RORα and β-catenin pathways, confirm that a nuclear receptor employs distinct coactivator complexes at different target genes, and provide a logic for early RORα expression in coordinating expression of genes required for reciprocal signals in cerebellar development

    What are the Effects of Severe Visual Impairment on the Cortical Organization and Connectivity of Primary Visual Cortex?

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    The organization and connections of the primary visual area (V1) were examined in mice that lacked functional rods (Gnat-/-), but had normal cone function. Because mice are nocturnal and rely almost exclusively on rod vision for normal behaviors, the Gnat-/- mice used in the present study are considered functionally blind. Our goal was to determine if visual cortex is reorganized in these mice, and to examine the neuroanatomical connections that may subserve reorganization. We found that most neurons in V1 responded to auditory, or some combination of auditory, somatosensory, and/or visual stimulation. We also determined that cortical connections of V1 in Gnat-/- mice were similar to those in normal animals, but even in normal animals, there is sparse input from auditory cortex (AC) to V1. An important observation was that most of the subcortical inputs to V1 were from thalamic nuclei that normally project to V1 such as the lateral geniculate (LG), lateral posterior (LP), and lateral dorsal (LD) nuclei. However, V1 also received some abnormal subcortical inputs from the anterior thalamic nuclei, the ventral posterior, the ventral lateral and the posterior nuclei. While the vision generated from the small number of cones appears to be sufficient to maintain most of the patterns of normal connectivity, the sparse abnormal thalamic inputs to VI, existing inputs from AC, and possibly abnormal inputs to LG and LP may be responsible for generating the alterations in the functional organization of V1

    Retinofugal Projections from Melanopsin-Expressing Retinal Ganglion Cells Revealed by Intraocular Injections of Cre-Dependent Virus

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    <div><p>To understand visual functions mediated by intrinsically photosensitive melanopsin-expressing retinal ganglion cells (mRGCs), it is important to elucidate axonal projections from these cells into the brain. Initial studies reported that melanopsin is expressed only in retinal ganglion cells within the eye. However, recent studies in <i>Opn4</i>-Cre mice revealed Cre-mediated marker expression in multiple brain areas. These discoveries complicate the use of melanopsin-driven genetic labeling techniques to identify retinofugal projections specifically from mRGCs. To restrict labeling to mRGCs, we developed a recombinant adeno-associated virus (AAV) carrying a Cre-dependent reporter (human placental alkaline phosphatase) that was injected into the vitreous of <i>Opn4</i>-Cre mouse eyes. The labeling observed in the brain of these mice was necessarily restricted specifically to retinofugal projections from mRGCs in the injected eye. We found that mRGCs innervate multiple nuclei in the basal forebrain, hypothalamus, amygdala, thalamus and midbrain. Midline structures tended to be bilaterally innervated, whereas the lateral structures received mostly contralateral innervation. As validation of our approach, we found projection patterns largely corresponded with previously published results; however, we have also identified a few novel targets. Our discovery of projections to the central amygdala suggests a possible direct neural pathway for aversive responses to light in neonates. In addition, projections to the accessory optic system suggest that mRGCs play a direct role in visual tracking, responses that were previously attributed to other classes of retinal ganglion cells. Moreover, projections to the zona incerta raise the possibility that mRGCs could regulate visceral and sensory functions. However, additional studies are needed to investigate the actual photosensitivity of mRGCs that project to the different brain areas. Also, there is a concern of "overlabeling" with very sensitive reporters that uncover low levels of expression. Light-evoked signaling from these cells must be shown to be of sufficient sensitivity to elicit physiologically relevant responses.</p></div

    Central targets of mRGCs in the brain.

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    <p>Representative coronal sections (rostral to caudal) from the brains of <i>Opn4</i><sup><i>cre</i></sup> mice with intravitreal injection of AAV-flex-plap into the right eye. Left panels in each row are low-power images of brain sections processed to visualize alkaline phosphate. The next panel in each row is an image of an adjacent brain section stained for Cytochrome Oxidase. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149501#pone.0149501.t001" target="_blank">Table 1</a> for nomenclature. Scale bar: 200 <b>μ</b>m (inset, 100 <b>μ</b>m).</p
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