Estimating the glutamate transporter surface density in distinct sub-cellular compartments of mouse hippocampal astrocytes.

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes, and previous estimates have been obtained about their surface expression in astrocytes of the rat hippocampus and cerebellum. Analogous estimates for the mouse hippocampus are currently not available. In this work, we derive the surface density of astrocytic glutamate transporters in mice of different ages via quantitative dot blot. We find that the surface density of glial glutamate transporters is similar in 7-8 week old mice and rats. In mice, the levels of glutamate transporters increase until about 6 months of age and then begin to decline slowly. Our data, obtained from a combination of experimental and modeling approaches, point to the existence of stark differences in the density of expression of glutamate transporters across different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on their level of synaptic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on how heterogeneity in the spatial distribution of glutamate transporters in the plasma membrane of hippocampal astrocytes my alter glutamate receptor activation out of the synaptic cleft.

Loss of the neurodevelopmental Joubert syndrome causing protein, Ahi1, causes motor and muscle development delays independent of central nervous system involvement.

Joubert syndrome (JBTS) is a predominantly autosomal recessive neurodevelopmental disorder that presents with characteristic malformations of the cerebellar vermis, superior cerebellar peduncles and midbrain in humans. Accompanying these malformations are a heterogeneous set of clinical symptoms, which frequently include deficits in motor and muscle function, such as hypotonia (low muscle tone) and ataxia (clumsiness). These symptoms are attributed to improper development of the hindbrain, but no direct evidence has been reported linking these in JBTS. Here, we describe muscle developmental defects in a mouse with a targeted deletion of the Abelson helper integration site 1 gene, Ahi1, one of the genes known to cause JBTS in humans. While FVB/NJ Ahi1-/- mice display no gross malformations of the cerebellum, deficits are observed in several measures of motor function, strength, and body development. Specifically, Ahi1-/- mice show delayed physical development, delays in surface reflex righting as neonates, and reductions in grip strength and spontaneous locomotor activity as adults. Additionally, Ahi1-/- mice showed evidence of muscle-specific contributions to this phenotype, such as reductions in 1) myoblast differentiation potential in vitro, 2) muscle desmin expression, and 3) overall muscle mass, myonuclear domain, and muscle fiber cross-sectional area. Together, these data suggest that loss of Ahi1 may cause abnormalities in the differentiation of myoblasts to mature muscle cells. Moreover, Ahi1 loss impacts muscle development directly, outside of any indirect impact of cerebellar malformations, revealing a novel myogenic cause for hypotonia in JBTS.

Anatomy and immunochemical characterization of the non-arterial peptidergic diffuse dural innervation of the rat and Rhesus monkey: Implications for functional regulation and treatment in migraine.

Objective The interplay between neuronal innervation and other cell types underlies the physiological functions of the dura mater and contributes to pathophysiological conditions such as migraine. We characterized the extensive, but understudied, non-arterial diffuse dural innervation (DDI) of the rat and Rhesus monkey. Methods We used a comprehensive integrated multi-molecular immunofluorescence labeling strategy to extensively profile the rat DDI and to a lesser extent that of the Rhesus monkey. Results The DDI was distributed across a dense, pervasive capillary network and included free nerve endings of peptidergic CGRP-expressing C fibers that were closely intertwined with noradrenergic (NA) sympathetic fibers and thin-caliber nonpeptidergic "C/Aδ" fibers. These newly identified C/Aδ fibers were unmyelinated, like C fibers, but expressed NF200, usually indicative of Aδ fibers, and uniquely co-labeled for the CGRP co-receptor, RAMP1. Slightly-larger caliber NF200-positive fibers co-labeled for myelin basic protein (MBP) and terminated as unbranched corpuscular endings. The DDI peptidergic fibers co-labeled for the lectin IB4 and expressed presumably excitatory α1-adrenergic receptors, as well as inhibitory 5HT1D receptors and the delta opioid receptor (δOR), but rarely the mu opioid receptor (µOR). Labeling for P2X3, TRPV1, TRPA1, and parasympathetic markers was not observed in the DDI. Interpretation These results suggest potential functional interactions, wherein peptidergic DDI fibers may be activated by stress-related sympathetic activity, resulting in CGRP release that could be detected in the circulation. CGRP may also activate nonpeptidergic C/Aδ fibers that are likely mechanosensitive or polymodal, leading to activation of post-synaptic pain transmission circuits. The distribution of α1-adrenergic receptors, RAMP1, and the unique expression of the δOR on CGRP-expressing DDI fibers suggest strategies for functional modulation and application to therapy.

MS AHI1 genetic risk promotes IFNγ+ CD4+ T cells.

OBJECTIVE: To study the influence of the Abelson helper integration site 1 (AHI1) locus associated with MS susceptibility on CD4+ T cell function. METHODS: We characterized the chromatin state of T cells in the MS-associated AHI1 linkage disequilibrium (LD) block. The expression and the role of the AHI1 variant were examined in T cells from genotyped healthy subjects who were recruited from the PhenoGenetic Project, and the function of AHI1 was explored using T cells from Ahi1 knockout mice. RESULTS: Chromatin state analysis reveals that the LD block containing rs4896153, which is robustly associated with MS susceptibility (odds ratio 1.15, p = 1.65 × 10-13), overlaps with strong enhancer regions that are present in human naive and memory CD4+ T cells. Relative to the rs4896153A protective allele, the rs4896153T susceptibility allele is associated with decreased AHI1 mRNA expression, specifically in naive CD4+ T cells (p = 1.73 × 10-74, n = 213), and we replicate this effect in an independent set of subjects (p = 2.5 × 10-9, n = 32). Functional studies then showed that the rs4896153T risk variant and the subsequent decreased AHI1 expression were associated with reduced CD4+ T cell proliferation and a specific differentiation into interferon gamma (IFNγ)-positive T cells when compared with the protective rs4896153A allele. This T cell phenotype was also observed in murine CD4+ T cells with genetic deletion of Ahi1. CONCLUSIONS: Our findings suggest that the effect of the AHI1 genetic risk for MS is mediated, in part, by enhancing the development of proinflammatory IFNγ+ T cells that have previously been implicated in MS and its mouse models.

BranchAnalysis2D/3D automates morphometry analyses of branching structures.

BACKGROUND: Morphometric analyses of biological features have become increasingly common in recent years with such analyses being subject to a large degree of observer bias, variability, and time consumption. While commercial software packages exist to perform these analyses, they are expensive, require extensive user training, and are usually dependent on the observer tracing the morphology. NEW METHOD: To address these issues, we have developed a broadly applicable, no-cost ImageJ plugin we call 'BranchAnalysis2D/3D', to perform morphometric analyses of structures with branching morphologies, such as neuronal dendritic spines, vascular morphology, and primary cilia. RESULTS: Our BranchAnalysis2D/3D algorithm allows for rapid quantification of the length and thickness of branching morphologies, independent of user tracing, in both 2D and 3D data sets. COMPARISON WITH EXISTING METHODS: We validated the performance of BranchAnalysis2D/3D against pre-existing software packages using trained human observers and images from brain and retina. We found that the BranchAnalysis2D/3D algorithm outputs results similar to available software (i.e., Metamorph, AngioTool, Neurolucida), while allowing faster analysis times and unbiased quantification. CONCLUSIONS: BranchAnalysis2D/3D allows inexperienced observers to output results like a trained observer but more efficiently, thereby increasing the consistency, speed, and reliability of morphometric analyses.