Kristen D. Phend

Techniques to optimize post-embedding single and double staining for amino acid neurotransmitters.

We report a number of technical refinements for single and double staining with post-embedding electron microscopy for glutamate, aspartate, and gamma-aminobutyric acid. Best results were obtained with 2.5% glutaraldehyde in the fixative and by minimizing the duration of plastic polymerization and the interval between cutting and reacting. Quantitative documentation of the ability of exogenous glutamate, aspartate, and gamma-aminobutyric acid to block their immune staining is provided. Increased intensity of staining with the glutamate and aspartate antisera resulted from preincubation of glutamate antiserum with aspartate and aspartate antiserum with glutamate. To perform double staining with antisera raised in the same species, it was necessary to block antigenicity of the first antiserum; best results were obtained with hot paraformaldehyde fumes. By using a detergent instead of etching, these methods permitted the simultaneous visualization of tracers to identify neuroanatomic pathways.

An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry.

Immunocytochemistry for amino acids with post-embedding gold is compatible with glutaraldehyde fixation, osmication, and embedding in epoxy-based plastics, but immunogold detection of larger molecules in the central nervous system commonly requires special procedures, e.g. minimizing exposure to glutaraldehyde, eliminating osmium, cryosectioning, and/or embedding in acrylic plastics. These make samples more difficult to prepare and view and may compromise structural preservation. We report a new technique, fixing with high levels of glutaraldehyde, replacing osmium with tannic acid followed by other heavy metals and p-phenylenediamine, and embedding in Epon. This method optimizes antigenicity while retaining the structural preservation and convenient handling of standard embedding techniques. Compared to standard Epon embedment, labeling for neuropeptides in brain and spinal cord is improved. Moreover, the present method yields excellent labeling of glutamate receptors (difficult to identify with traditional post-embedding techniques) and enables simultaneous visualization of associated neurotransmitters.

EM colocalization of AMPA and NMDA receptor subunits at synapses in rat cerebral cortex

Electrophysiology and light microscopy suggest that a single excitatory synapse may use both amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors. Using immunogold electron microscopy, we here provide direct evidence for colocalization at individual synapses in sensorimotor cortex of adult rats. Colocalization was most commonly observed on dendritic spines; subunits of the two classes of receptors seemed to be independently distributed within the synaptic active zone.

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 brains 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.

Primary afferent terminals that express presynaptic NR1 in rats are mainly from myelinated, mechanosensitive fibers.

Presynaptic N‐methyl‐D‐aspartate (NMDA) receptors in terminals of primary afferents to spinal cord of rats were first reported by Liu et al. (1994; Proc. Natl. Acad. Sci. USA 91:8383–8387) and were proposed to modulate nociceptive input (Liu et al. [1997] Nature 386:721–724). We previously demonstrated kainate and AMPA receptors in numerous primary afferent terminals in the spinal cord fixed with diluted paraformaldehyde and no glutaraldehyde. Therefore, we reinvestigated the occurrence of presynaptic NMDAR1 (NR1) with this fixation protocol. With confocal microscopy, numerous immunofluorescent puncta were double‐stained for NR1 and the presynaptic marker synaptophysin throughout the spinal gray. NR1‐immunostained puncta costained more frequently with a tracer that labels myelinated afferents (cholera toxin subunit B; CTB) than with a tracer that labels non‐peptidergic unmyelinated afferents (Griffonia simplicifolia isolectin B4; IB4). Virtually no double staining was found for NR1 and calcitonin gene‐related peptide (CGRP), which labels somatic peptidergic primary afferents. In the gracile nucleus, virtually all puncta labeled for CTB appeared immunopositive for NR1. At the electron microscopic level, most immunopositive terminals in spinal cord and gracile nucleus displayed morphological characteristics of endings of myelinated primary afferents. NR1 was presynaptic in 60–65% of all synapses in which it was expressed pre‐ or postsynaptically, or both, in spinal laminae I–IV. Estimates for the gracile nucleus were higher (80%). No presynaptic NR1 was found in the ventroposterior thalamus. Because of the relative sparsity of presynaptic NR1 in terminals in laminae I and IIo and in terminals of peptidergic unmyelinated afferents, it is suggested that presynaptic NMDA receptors play a more significant role in modulation of mechanosensitive, innocuous input than in nociception. J. Comp. Neurol. 460:191–202, 2003.

A new double-labeling method demonstrates transmitter-specific projections

We report a method which combines retrograde transport of the fluorescent dye, diamidino yellow dihydrochloride (DY), with peroxidase immunocytochemical staining for glutamic acid decarboxylase (GAD), an enzyme essential for the synthesis of gamma-aminobutyric acid (GABA). Cells exhibiting both retrograde fluorescent label and GAD-positive immunoreactivity were observed in the cerebellar cortex, the striatum and the ventrobasal complex following injections of DY into the superior vestibular nucleus, substantia nigra and dorsal thalamus. The method, which can in principle be applied to any antigen, takes advantage of the differential nuclear/cytoplasmic distribution of the two stains. By using appropriate filter combinations and balanced epi- and transillumination, double-labeled cells are readily identifiable.

Subcellular organization of UBE3A in neurons

Ubiquitination regulates a broad array of cellular processes, and defective ubiquitination is implicated in several neurological disorders. Loss of the E3 ubiquitin–protein ligase UBE3A causes Angelman syndrome. Despite its clinical importance, the normal role of UBE3A in neurons is still unclear. As a step toward deciphering its possible functions, we performed high‐resolution light and electron microscopic immunocytochemistry. We report a broad distribution of UBE3A in neurons, highlighted by concentrations in axon terminals and euchromatin‐rich nuclear domains. Our findings suggest that UBE3A may act locally to regulate individual synapses while also mediating global, neuronwide influences through the regulation of gene transcription. J. Comp. Neurol. 525:233–251, 2017.

Organization of TNIK in dendritic spines.

Tumor necrosis factor receptor‐associated factor 2 (TRAF2)‐ and noncatalytic region of tyrosine kinase (NCK)‐interacting kinase (TNIK) has been identified as an interactor in the psychiatric risk factor, Disrupted in Schizophrenia 1 (DISC1). As a step toward deciphering its function in the brain, we performed high‐resolution light and electron microscopic immunocytochemistry. We demonstrate here that TNIK is expressed in neurons throughout the adult mouse brain. In striatum and cerebral cortex, TNIK concentrates in dendritic spines, especially in the vicinity of the lateral edge of the synapse. Thus, TNIK is highly enriched at a microdomain critical for glutamatergic signaling. J. Comp. Neurol. 523:1913–1924, 2015

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

In the presence of netrin, tripartite motif protein 9 (TRIM9) promotes deleted in colorectal cancer (DCC) clustering, but TRIM9-dependent ubiquitination of DCC is reduced. Loss of ubiquitination promotes an interaction between DCC and FAK and FAK activation. FAK activation is required for the progression from SNARE assembly to exocytic vesicle fusion, which supplies membrane material for axon branching.

Conjugate Immunofluorescence—SEM Array Tomography for Studying Mammalian Synapses and Axons

Conjugate immunofluorescence—SEM array tomography enables the imaging of both the molecular content and the ultrastructure of tissues. The method is based on physical ultrathin serial sectioning, immunostaining and acquiring fluorescence and electron microscopy images of resin embedded tissues, followed by computational volume reconstruction and analysis. Conjugate immunofluorescence—SEM array tomography has been used for the study of brain tissue, and in particular for the characterization of diverse synapses and axons.