Primary Afferent Terminals in Spinal Cord Express Presynaptic AMPA Receptors

Larger dorsal root ganglion neurons are stained by an antibody for the C terminus of glutamate receptor subunit 2 (GluR2) and GluR3 (GluR2/3) rather than by an antibody for GluR4. In dorsal roots, anti-GluR2/3 stains predominantly myelinated fibers; anti-GluR4 or anti-GluR2/4 stains predominantly unmyelinated fibers. In the dorsal horn, puncta immunopositive for synaptophysin and GluR2/3 are predominantly in laminas III and IV, whereas puncta immunopositive for synaptophysin and GluR4 or GluR2/4 are predominantly in laminas I and II. Puncta immunopositive for GluR2/3 costain with the B subunit of cholera toxin, whereas puncta immunopositive for GluR2/4 costain with isolectin B4 after injections of these tracers in the sciatic nerve. No puncta costain with calcitonin gene-related peptide and AMPA receptor subunits. Electron microscopy indicates that AMPA receptor-immunopositive terminals are more numerous than suggested by confocal microscopy. Of all synapses in which immunostaining is presynaptic, postsynaptic, or both, the percentage of presynaptic immunostain is approximately 70% with anti-GluR4 or anti-GluR2/4 (in laminas I-III), 25-30% with anti-GluR2/3 (in laminas III and IV), and 5% with anti-GluR2 (in laminas I-III). Because of fixation constraints, the types of immunostained terminals could be identified only on the basis of morphological characteristics. Many terminals immunostained for GluR2/3, GluR4, or GluR2/4 have morphological features of endings of primary afferents. Terminals with morphological characteristics of presumed GABAergic terminals are also immunostained with anti-GluR2/4 and anti-GluR4 in laminas I and II and with anti-GluR2/3 in laminas III and IV. The conspicuous and selective expression of presynaptic AMPA receptor subunits may contribute to the characteristic physiological profile of different classes of primary afferents and suggests an important mechanism for the modulation of transmitter release by terminals of both myelinated and unmyelinated primary afferents

TRIM9-dependent ubiquitination of DCC constrains kinase signaling, exocytosis, and axon branching

Extracellular netrin-1 and its receptor deleted in colorectal cancer (DCC) promote axon branching in developing cortical neurons. Netrin-dependent morphogenesis is preceded by multimerization of DCC, activation of FAK and Src family kinases, and increases in exocytic vesicle fusion, yet how these occurrences are linked is unknown. Here we demonstrate that tripartite motif protein 9 (TRIM9)-dependent ubiquitination of DCC blocks the interaction with and phosphorylation of FAK. Upon netrin-1 stimulation TRIM9 promotes DCC multimerization, but TRIM9-dependent ubiquitination of DCC is reduced, which promotes an interaction with FAK and subsequent FAK activation. We found that inhibition of FAK activity blocks elevated frequencies of exocytosis in vitro and elevated axon branching in vitro and in vivo. Although FAK inhibition decreased soluble N-ethylmaleimide attachment protein receptor (SNARE)-mediated exocytosis, assembled SNARE complexes and vesicles adjacent to the plasma membrane increased, suggesting a novel role for FAK in the progression from assembled SNARE complexes to vesicle fusion in developing murine neurons

Neurocan Inhibits Semaphorin 3F Induced Dendritic Spine Remodeling Through NrCAM in Cortical Neurons

Neurocan is a chondroitin sulfate proteoglycan present in perineuronal nets, which are associated with closure of the critical period of synaptic plasticity. During postnatal development of the neocortex dendritic spines on pyramidal neurons are initially overproduced; later they are pruned to achieve an appropriate balance of excitatory to inhibitory synapses. Little is understood about how spine pruning is terminated upon maturation. NrCAM (Neuron-glial related cell adhesion molecule) was found to mediate spine pruning as a subunit of the receptor complex for the repellent ligand Semaphorin 3F (Sema3F). As shown here in the postnatal mouse frontal and visual neocortex, Neurocan was localized at both light and electron microscopic level to the cell surface of cortical pyramidal neurons and was adjacent to neuronal processes and dendritic spines. Sema3F-induced spine elimination was inhibited by Neurocan in cortical neuron cultures. Neurocan also blocked Sema3F-induced morphological retraction in COS-7 cells, which was mediated through NrCAM and other subunits of the Sema3F holoreceptor, Neuropilin-2, and PlexinA3. Cell binding and ELISA assays demonstrated an association of Neurocan with NrCAM. Glycosaminoglycan chain interactions of Neurocan were required for inhibition of Sema3F-induced spine elimination, but the C-terminal sushi domain was dispensable. These results describe a novel mechanism wherein Neurocan inhibits NrCAM/Sema3F-induced spine elimination

Mapping Synapses by Conjugate Light-Electron Array Tomography

Synapses of the mammalian CNS are diverse in size, structure, molecular composition, and function. Synapses in their myriad variations are fundamental to neural circuit development, homeostasis, plasticity, and memory storage. Unfortunately, quantitative analysis and mapping of the brain's heterogeneous synapse populations has been limited by the lack of adequate single-synapse measurement methods. Electron microscopy (EM) is the definitive means to recognize and measure individual synaptic contacts, but EM has only limited abilities to measure the molecular composition of synapses. This report describes conjugate array tomography (AT), a volumetric imaging method that integrates immunofluorescence and EM imaging modalities in voxel-conjugate fashion. We illustrate the use of conjugate AT to advance the proteometric measurement of EM-validated single-synapse analysis in a study of mouse cortex