GABA Expression and Regulation by Sensory Experience in the Developing Visual System

The developing retinotectal system of the Xenopus laevis tadpole is a model of choice for studying visual experience-dependent circuit maturation in the intact animal. The neurotransmitter gamma-aminobutyric acid (GABA) has been shown to play a critical role in the formation of sensory circuits in this preparation, however a comprehensive neuroanatomical study of GABAergic cell distribution in the developing tadpole has not been conducted. We report a detailed description of the spatial expression of GABA immunoreactivity in the Xenopus laevis tadpole brain at two key developmental stages: stage 40/42 around the onset of retinotectal innervation and stage 47 when the retinotectal circuit supports visually-guided behavior. During this period, GABAergic neurons within specific brain structures appeared to redistribute from clusters of neuronal somata to a sparser, more uniform distribution. Furthermore, we found that GABA levels were regulated by recent sensory experience. Both ELISA measurements of GABA concentration and quantitative analysis of GABA immunoreactivity in tissue sections from the optic tectum show that GABA increased in response to a 4 hr period of enhanced visual stimulation in stage 47 tadpoles. These observations reveal a remarkable degree of adaptability of GABAergic neurons in the developing brain, consistent with their key contributions to circuit development and function

Distribution of GABA immunoreactivity in stage 42 and stage 47 tadpole CNS.

<p>A. Stage 42: Schematic (left) indicating relative positions of montaged sagittal sections of the tadpole brain. Blue is the cell body area; white is the neuropil area. Sections (1–4) show GABA immunostaining (green) counterstained with the nuclear label, propidium iodide (PI, blue). GABA staining alone is presented in the right panels (1′–4′). In panels 1′–4′ <i>arrowheads</i> indicate GABA containing somata, <i>filled arrows</i> are GABA-positive axon tracts, and <i>open arrows</i> denote GABA-sparse zones. B. Sagittal series through a stage 47 tadpole brain. The pattern of GABA-immunoreactivity in the brain is similar to stage 42 except for a dispersion of the dense clusters of GABA immunoreactive cells seen in younger brains and the vast expansion of the labeled cell body regions, neuropil and axon tracts in the older tadpoles. Scale bars, 250 µm. See text for details.</p

Quantification of immunofluorescence intensity for ßIII-tubulin, GABA and the GABA/ßIII-tubulin ratio, measured in tectal neuropil and the cell body layer from animals exposed to visual stimulus or dark.

<p>Fluorescence intensity values are in arbitrary units.</p

GABA immunoreactivity in optic tectum of stage 42 and stage 47 tadpoles (horizontal plane).

<p>A. Stage 42: Left: Schematic indicating relative positions of horizontal sections through the dorsal midbrain and locations of major brain regions (top left). Schematic of a horizontal section through the brain with locations of brain regions labeled. Blue is the cell body area; white is the neuropil area. An image of a GABA-immunolabeled right hemisection is superimposed on the schematic (bottom left). Sections (1–5) show GABA-immunoreactivity (green) counterstained with PI (blue). B. Schematic of horizontal brain section (left) showing regions of high magnification images, shown to the right. Higher magnification single optical sections from stage 42 midbrain. B1. Intense GABA immunolabeling of axons in the tecto-tegmental commissure (ttc) and posterior commissure (pc) (solid arrows). B2. Clustered GABA-immunoreactive neurons in the optic tectum (solid arrows) extend processes toward the neuropil. B2a, b. Enlargements of boxed regions in B2 showing GABA-immunoreactive processes (arrows) extending from labeled cell bodies (arrowheads in 2a,b). C. Stage 47: Left. Schematics comparable to A. Sections (1–5) of GABA-immunoreactivity (green) and PI counterstain (blue). GABA-immunoreactivity becomes more broadly distributed across the optic tectal cell body layer (arrowheads) and neuropil. D. Higher magnification (single optical sections) showing strong GABA labeling in the lateral forebrain bundle (lfb, D1), and sparse GABA-positive somata in the caudolateral optic tectum (D2a, arrowheads) extending GABA-positive processes toward the neuropil (D2a, solid arrows). D3. The border between the caudal optic tectum and the medial hindbrain (HB) shows that the proliferative zone in caudal tectum is negative for GABA immunostaining (arrows), whereas neuronal cell bodies and processes in the medial HB are GABA-immunolabeled (arrowheads). Scale bars, A, C: 50 µm; B1, 2: 20 µm; B2a,b: 10 µm: D1,3: 30 µm; D2a,b: 20 µm.</p

GABA immunoreactvity in retina of stage 42 and stage 47 tadpoles.

<p>A. Stage 42 coronal cryosection of the retina showing GABA immunoreactivity (green) and propidium iodide (blue) staining in the retina. B. GABA immunolabeling alone. At this stage, GABA immunolabeling is absent from the ganglion cell layer (GCL, hollow arrow) and outer nuclear label (ONL, hollow arrow). GABA-positive cell somata are densely packed in the INL and ramify processes into the GCL (solid arrow) and OPL (solid arrow). D. Stage 47 sections through retina showing GABA immunoreactivity (green) and propidium iodide counterstain (blue). A few GABA-positive somata are now evident in the GCL (arrowheads). GABA immunoreactivity is present in the IPL, INL and OPL, but absent in the ONL. Scale bar, 25 µm.</p

Quantification of GABA levels (µg/ml) by ELISA.

<p>Tissue was collected from animals exposed to visual stimulation or kept in the dark. A tissue homogenate sample was divided into 4 aliquots and each aliquot was analyzed separately.</p

GABA immunoreactivity in optic tectum of stage 42 and stage 47 tadpoles (coronal plane).

<p>A. Stage 42: Left: Schematic indicating relative positions of coronal sections through the midbrain. Right: Sections (1–4) show GABA immunoreactivity (green) with PI counterstain (blue). Schematics under each section identify major brain regions in the sections. Blue is the cell body area; white is the neuropil area. GABA-positive cells are clustered medially in the anterior tectum (arrowheads) and send processes to the neuropil (solid arrows; section 1). A cluster of GABA-labeled neurons extends from the anterior ventricular region posteriorly and laterally within the tectum (sections 1–3, arrowheads). GABA-positive neurons are dispersed in caudal tectum (section 4, arrowhead). The tegmentum of stage 42 tadpoles has relatively few GABA-immunoreactive neurons (open arrows, sections 1–4), but extensive GABA-immunoreactivity in the lateral neuropil. B. Stage 47: Schematics shown are comparable to those in A. GABA-positive cells are interspersed throughout the optic tectum dorsally and in the tegmentum. The labeled neurons are distributed more laterally than in the younger tadpoles (arrowheads; sections 1–4). The zone closest to the tectal ventricle is largely devoid of GABA-immunoreactivity (section 2, hollow arrow). The tectal and tegmental neuropil regions are intensely GABA immunoreactive. Scale bar, 50 µm.</p

Profiles of Extracellular miRNA in Cerebrospinal Fluid and Serum from Patients with Alzheimer's and Parkinson's Diseases Correlate with Disease Status and Features of Pathology

<div><p>The discovery and reliable detection of markers for neurodegenerative diseases have been complicated by the inaccessibility of the diseased tissue- such as the inability to biopsy or test tissue from the central nervous system directly. RNAs originating from hard to access tissues, such as neurons within the brain and spinal cord, have the potential to get to the periphery where they can be detected non-invasively. The formation and extracellular release of microvesicles and RNA binding proteins have been found to carry RNA from cells of the central nervous system to the periphery and protect the RNA from degradation. Extracellular miRNAs detectable in peripheral circulation can provide information about cellular changes associated with human health and disease. In order to associate miRNA signals present in cell-free peripheral biofluids with neurodegenerative disease status of patients with Alzheimer's and Parkinson's diseases, we assessed the miRNA content in cerebrospinal fluid and serum from postmortem subjects with full neuropathology evaluations. We profiled the miRNA content from 69 patients with Alzheimer's disease, 67 with Parkinson's disease and 78 neurologically normal controls using next generation small RNA sequencing (NGS). We report the average abundance of each detected miRNA in cerebrospinal fluid and in serum and describe 13 novel miRNAs that were identified. We correlated changes in miRNA expression with aspects of disease severity such as Braak stage, dementia status, plaque and tangle densities, and the presence and severity of Lewy body pathology. Many of the differentially expressed miRNAs detected in peripheral cell-free cerebrospinal fluid and serum were previously reported in the literature to be deregulated in brain tissue from patients with neurodegenerative disease. These data indicate that extracellular miRNAs detectable in the cerebrospinal fluid and serum are reflective of cell-based changes in pathology and can be used to assess disease progression and therapeutic efficacy.</p></div

Ordinal regression analysis reveals miRNAs with progressive expression trends across increasing neurofibrillary tangle density.

<p>(<b>A</b>) We plotted four miRNAs (miR-181b-5p, miR-181d, miR-181a-5p and miR-9-3p) detected in CSF from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094839#pone-0094839-t005" target="_blank">Table? 5</a>. (<b>B</b>) miR-7i-3p and miR-10a-5p were selected from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094839#pone-0094839-t005" target="_blank">Table? 5</a>, significant for neurofibrillary tangle stage regression analysis in SER.</p

Differentially expressed miRNAs detected in the cerebrospinal fluid (CSF).

<p>Sample size for cerebrospinal fluid consisted of 62 AD, 57 PD and 65 control subjects. Results were filtered at adjusted p-value <0.05. The logarithmic base 2 fold change (FC) is relative to the first listed group for each comparison. Significant miRNAs were reported if their normalized base average is greater than 5 mapped reads and 0.7< FC(log2) or FC(log2) <−0.7. Superscript 1 indicates miRNAs that are differentially expressed in both patients with Alzheimer's disease and Parkinson's disease compared to control subjects. Superscript 2 indicates differentially expressed miRNAs in both CSF and SER biofluids for the corresponding analysis. Significant miRNAs with a superscript 3 are in low abundance, with normalized mean <10 mapped reads.</p