AB5905 Sigma-AldrichAnti-Vesicular Glutamate Transporter 1 Antibody

Mounting evidence suggests that neonatal tissue damage evokes alterations in spinal pain reflexes which persist into adulthood. However, less is known about potential concomitant effects on the transmission of nociceptive information to the brain, as the degree to which early injury modulates synaptic integration and membrane excitability in mature spinal projection neurons remains unclear. Here we demonstrate that neonatal surgical injury leads to a significant shift in the balance between synaptic excitation and inhibition onto identified lamina I projection neurons of the adult mouse spinal cord. The strength of direct primary afferent input to mature spino-parabrachial neurons was enhanced following neonatal tissue damage, whereas the efficacy of both GABAergic and glycinergic inhibition onto the same population was compromised. This was accompanied by reorganization in the pattern of sensory input to adult projection neurons, which included a greater prevalence of monosynaptic input from low-threshold A-fibers when preceded by early tissue damage. In addition, neonatal incision resulted in greater primary afferent-evoked action potential discharge in mature projection neurons. Overall, these results demonstrate that tissue damage during early life causes a long-term increase in the gain of spinal nociceptive circuits, and suggest that the prolonged consequences of neonatal trauma may not be restricted to the spinal cord but rather include excessive ascending signaling to supraspinal pain centers.

RET can be activated in cis or trans by its co-receptors and ligands in vitro, but the physiological roles of trans signaling are unclear. Rapidly adapting (RA) mechanoreceptors in dorsal root ganglia (DRGs) express Ret and the co-receptor Gfrα2 and depend on Ret for survival and central projection growth. Here, we show that Ret and Gfrα2 null mice display comparable early central projection deficits, but Gfrα2 null RA mechanoreceptors recover later. Loss of Gfrα1, the co-receptor implicated in activating RET in trans, causes no significant central projection or cell survival deficit, but Gfrα1;Gfrα2 double nulls phenocopy Ret nulls. Finally, we demonstrate that GFRα1 produced by neighboring DRG neurons activates RET in RA mechanoreceptors. Taken together, our results suggest that trans and cis RET signaling could function in the same developmental process and that the availability of both forms of activation likely enhances but not diversifies outcomes of RET signaling.

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.

NMDA receptors (NMDARs) play an essential role in some forms of synaptic plasticity, learning, and memory. Therefore, these receptors are highly regulated with respect to their localization, activation, and abundance both within and on the surface of mammalian neurons. Fundamental questions remain, however, regarding how this complex regulation is achieved. Using cell-based models and F-box Only Protein 2 (Fbxo2) knock-out mice, we found that the ubiquitin ligase substrate adaptor protein Fbxo2, previously reported to facilitate the degradation of the NMDAR subunit GluN1 in vitro, also functions to regulate GluN1 and GluN2A subunit levels in the adult mouse brain. In contrast, GluN2B subunit levels are not affected by the loss of Fbxo2. The loss of Fbxo2 results in greater surface localization of GluN1 and GluN2A, together with increases in the synaptic markers PSD-95 and Vglut1. These synaptic changes do not manifest as neurophysiological differences or alterations in dendritic spine density in Fbxo2 knock-out mice, but result instead in increased axo-dendritic shaft synapses. Together, these findings suggest that Fbxo2 controls the abundance and localization of specific NMDAR subunits in the brain and may influence synapse formation and maintenance.

The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.

Human adolescence is a crucial stage of neurological development during which ethanol (EtOH) consumption is often at its highest. Alcohol abuse during adolescence may render individuals at heightened risk for subsequent alcohol abuse disorders, cognitive dysfunction, or other neurological impairments by irreversibly altering long-term brain function. To test this possibility, we modeled adolescent alcohol abuse (i.e., intermittent EtOH exposure during adolescence [AIE]) in rats to determine whether adolescent exposure to alcohol leads to long-term structural and functional changes that are manifested in adult neuronal circuitry.We specifically focused on hippocampal area CA1, a brain region associated with learning and memory. Using electrophysiological, immunohistochemical, and neuroanatomical approaches, we measured post-AIE changes in synaptic plasticity, dendritic spine morphology, and synaptic structure in adulthood.We found that AIE-pretreated adult rats manifest robust long-term potentiation, induced at stimulus intensities lower than those required in controls, suggesting a state of enhanced synaptic plasticity. Moreover, AIE resulted in an increased number of dendritic spines with characteristics typical of immaturity. Immunohistochemistry-based analysis of synaptic structures indicated a significant decrease in the number of co-localized pre- and postsynaptic puncta. This decrease is driven by an overall decrease in 2 postsynaptic density proteins, PSD-95 and SAP102.Taken together, these findings reveal that repeated alcohol exposure during adolescence results in enduring structural and functional abnormalities in the hippocampus. These synaptic changes in the hippocampal circuits may help to explain learning-related behavioral changes in adult animals preexposed to AIE.

Autism is a multifactorial neurodevelopmental disorder affecting more males than females; consequently, under a multifactorial genetic hypothesis, females are affected only when they cross a higher biological threshold. We hypothesize that deleterious variants at conserved residues are enriched in severely affected patients arising from female-enriched multiplex families with severe disease, enhancing the detection of key autism genes in modest numbers of cases. Here we show the use of this strategy by identifying missense and dosage sequence variants in the gene encoding the adhesive junction-associated δ-catenin protein (CTNND2) in female-enriched multiplex families and demonstrating their loss-of-function effect by functional analyses in zebrafish embryos and cultured hippocampal neurons from wild-type and Ctnnd2 null mouse embryos. Finally, through gene expression and network analyses, we highlight a critical role for CTNND2 in neuronal development and an intimate connection to chromatin biology. Our data contribute to the understanding of the genetic architecture of autism and suggest that genetic analyses of phenotypic extremes, such as female-enriched multiplex families, are of innate value in multifactorial disorders.

Leucine-rich repeat kinase 2 (LRRK2) is the single most common genetic cause of both familial and sporadic Parkinson's disease (PD), both of which share pathogenetic and neurologic similarities with human immunodeficiency virus 1 (HIV-1)-associated neurocognitive disorders (HAND). Pathologic LRRK2 activity may also contribute to neuroinflammation, because microglia lacking LRRK2 exposed to proinflammatory stimuli have attenuated responses. Because microglial activation is a hallmark of HIV-1 neuropathology, we have investigated the role of LRRK2 activation using in vitro and in vivo models of HAND. We hypothesize that LRRK2 is a key modulator of microglial inflammatory responses, which play a pathogenic role in both HAND and PD, and that these responses may cause or exacerbate neuronal damage in these diseases. The HIV-1 Tat protein is a potent neurotoxin produced during HAND that induces activation of primary microglia in culture and long-lasting neuroinflammation and neurotoxicity when injected into the CNS of mice. We found that LRRK2 inhibition attenuates Tat-induced pS935-LRRK2 expression, proinflammatory cytokine and chemokine expression, and phosphorylated p38 and Jun N-terminal kinase signaling in primary microglia. In our murine model, cortical Tat injection in LRRK2 knock-out (KO) mice results in significantly diminished neuronal damage, as assessed by microtubule-associated protein 2 (MAP2), class III β-tubulin TUJ1, synapsin-1, VGluT, and cleaved caspase-3 immunostaining. Furthermore, Tat-injected LRRK2 KO animals have decreased infiltration of peripheral neutrophils, and the morphology of microglia from these mice were similar to that of vehicle-injected controls. We conclude that pathologic activation of LRRK2 regulates a significant component of the neuroinflammation associated with HAND.

Autoimmune-mediated anti-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) encephalitis is a severe but treatment-responsive disorder with prominent short-term memory loss and seizures. The mechanisms by which patient antibodies affect synapses and neurons leading to symptoms are poorly understood.The effects of patient antibodies on cultures of live rat hippocampal neurons were determined with immunostaining, Western blot, and electrophysiological analyses.We show that patient antibodies cause a selective decrease in the total surface amount and synaptic localization of GluA1- and GluA2-containing AMPARs, regardless of receptor subunit binding specificity, through increased internalization and degradation of surface AMPAR clusters. In contrast, patient antibodies do not alter the density of excitatory synapses, N-methyl-D-aspartate receptor (NMDAR) clusters, or cell viability. Commercially available AMPAR antibodies directed against extracellular epitopes do not result in a loss of surface and synaptic receptor clusters, suggesting specific effects of patient antibodies. Whole-cell patch clamp recordings of spontaneous miniature postsynaptic currents show that patient antibodies decrease AMPAR-mediated currents, but not NMDAR-mediated currents. Interestingly, several functional properties of neurons are also altered: inhibitory synaptic currents and vesicular γ-aminobutyric acid transporter (vGAT) staining intensity decrease, whereas the intrinsic excitability of neurons and short-interval firing increase.These results establish that antibodies from patients with anti-AMPAR encephalitis selectively eliminate surface and synaptic AMPARs, resulting in a homeostatic decrease in inhibitory synaptic transmission and increased intrinsic excitability, which may contribute to the memory deficits and epilepsy that are prominent in patients with this disorder.

While photoreceptor loss is the most devastating result of inherited retinal degenerations such as retinitis pigmentosa, inner retinal neurons also undergo significant alteration. Detailing these changes has become important as many vision restorative therapies target the remaining neurons. In this study, the rd1-Fos-Tau-LacZ (rd1-FTL) mouse model was used to explore inner retinal change at a late stage of retinal degeneration, after the loss of photoreceptor nuclei. The rd1-FTL model carries a mutation in the phosphodiesterase gene, Pde6b, and an axonally targeted transgenic beta galactosidase reporter system under the control of the c-fos promoter. Retinae of transgenic rd1-FTL mice and control FTL animals aged 2-12 months were processed for indirect fluorescence immunocytochemistry. At 2 months of age, a time when the majority of photoreceptor nuclei are lost, there was negligible c-fos reporter (FTL) expression, however, from 4 months, reporter expression was observed to increase within subpopulations of amacrine and ganglion cells within the central retina. These areas of inner retinal FTL expression coincided with regions that contained aberrant Müller cells. Specifically, these cells exhibited reduced glutamine synthetase and Kir4.1 immunolabelling, whilst showing evidence of proliferative gliosis (increased cyclinD1 and glial fibrillary acidic protein expression). These changes were limited to distinct regions where cone photoreceptor terminals were absent. Overall, these results highlight that distinct areas of the rd1-FTL central retina undergo significant glial alterations after cone photoreceptor loss. These areas coincide with up-regulation of the c-fos reporter in the inner retina, which may represent a change in neuronal function/plasticity. The rd1-FTL mouse is a useful model system to probe changes that occur in the inner retina at later stages of retinal degeneration.