Expression of the Excitatory Postsynaptic Scaffolding Protein, Shank3, in Human Brain: Effect of Age and Alzheimer’s Disease

Shank3 is a postsynaptic scaffolding protein of excitatory synapses. Mutations or variations of SHANK3 are associated with various psychiatric and neurological disorders. We set to determine its normal expression pattern in the human brain, and its change, if any, with age and Alzheimer’s disease (AD)-type β-amyloid (Aβ) and Tau pathogenesis. In general, Shank3 immunoreactivity (IR) exhibited largely a neuropil pattern with differential laminar/regional distribution across brain regions. In youth and adults, subsets of pyramidal/multipolar neurons in the cerebrum, striatum, and thalamus showed moderate IR, while some large-sized neurons in the brainstem and the granule cells in the cerebellar cortex exhibited light IR. In double immunofluorescence, Shank3 IR occurred at the sublemmal regions in neuronal somata and large dendrites, apposing to synaptophysin-labeled presynaptic terminals. In aged cases, immunolabeled neuronal somata were reduced, with disrupted neuropil labeling seen in the molecular layer of the dentate gyrus in AD cases. In immunoblot, levels of Shank3 protein were positively correlated with that of the postsynaptic density protein 95 (PSD95) among different brain regions. Levels of Shank3, PSD95, and synaptophysin immunoblotted in the prefrontal, precentral, and cerebellar cortical lysates were reduced in the aged and AD relative to youth and adult groups. Taken together, the differential Shank3 expression among brain structures/regions indicates the varied local density of the excitatory synapses. The enriched Shank3 expression in the forebrain subregions appears inconsistent with a role of this protein in the modulation of high cognitive functions. The decline of its expression in aged and AD brains may relate to the degeneration of excitatory synapses.

Accurate Neuronal Soma Segmentation Using 3D Multi-Task Learning U-Shaped Fully Convolutional Neural Networks

Neuronal soma segmentation is a crucial step for the quantitative analysis of neuronal morphology. Automated neuronal soma segmentation methods have opened up the opportunity to improve the time-consuming manual labeling required during the neuronal soma morphology reconstruction for large-scale images. However, the presence of touching neuronal somata and variable soma shapes in images brings challenges for automated algorithms. This study proposes a neuronal soma segmentation method combining 3D U-shaped fully convolutional neural networks with multi-task learning. Compared to existing methods, this technique applies multi-task learning to predict the soma boundary to split touching somata, and adopts U-shaped architecture convolutional neural network which is effective for a limited dataset. The contour-aware multi-task learning framework is applied to the proposed method to predict the masks of neuronal somata and boundaries simultaneously. In addition, a spatial attention module is embedded into the multi-task model to improve neuronal soma segmentation results. The Nissl-stained dataset captured by the micro-optical sectioning tomography system is used to validate the proposed method. Following comparison to four existing segmentation models, the proposed method outperforms the others notably in both localization and segmentation. The novel method has potential for high-throughput neuronal soma segmentation in large-scale optical imaging data for neuron morphology quantitative analysis.

Amyloid β and tau pathology in brains of aged pinniped species (sea lion, seal, and walrus)

Alzheimer’s disease (AD) is characterized by the accumulation of amyloid-β (Aβ) as senile plaques and cerebral amyloid angiopathy, and hyperphosphorylated tau (hp-tau) as neurofibrillary tangles in the brain. The AD-related pathology has been reported in several non-human animals, and most animals develop only the Aβ or tau pathology. We herein describe the Aβ and hp-tau pathology in the brains of aged pinniped species (seal, sea lion, and walrus). Molecular analyses revealed that the sequence of pinniped Aβ was identical to that of human Aβ. Histopathological examinations detected argyrophilic plaques composed of Aβ associated with dystrophic neurites in the cerebral cortex of aged pinnipeds. Astrogliosis and microglial infiltration were identified around Aβ plaques. Aβ deposits were observed in the blood vessel walls of the meninges and cerebrum. Pinniped tau protein was physiologically subjected to alternative splicing at exons 2, 3, and 10, and presented as five isoforms: two 3-repeat tau isoforms (1N3R, 2N3R) and three 4-repeat tau isoforms (0N4R, 1N4R, 2N4R); 0N3R tau isoform was absent. Histopathological examinations revealed argyrophilic fibrillar aggregates composed of hp-tau in the neuronal somata and neurites of aged pinniped brains. Few hp-tau aggregates were found in oligodendrocytes and microglia. Biochemically, hp-tau of the 3-repeat and 4-repeat isoforms was detected in brain sarkosyl-insoluble fractions. Aβ and hp-tau both predominantly accumulated in the neocortex, particularly the frontal cortex. Furthermore, the activation of GSK-3β was detected within cells containing hp-tau aggregates, and activated GSK-3β was strongly expressed in cases with severe hp-tau pathologies. The present results suggest that, in association with Aβ deposition, the activation of GSK-3β contributes to hp-tau accumulation in pinniped brains. Here, we report that pinniped species naturally accumulate Aβ and tau with aging, similar to the human AD pathology.

A Few TH-Immunoreactive Neurons Closely Appose DMX-Located Neuronal Somata Projecting to the Stomach Prepyloric Region in the Pig

The vagus nerve is responsible for efferent innervation and functional control of stomach functions. The efferent fibers originate from neurons located in the dorsal motor nucleus of the vagus (DMX) and undergo functional control of the local neuroregulatory terminals. The aim of the present study was to examine the existence of morphological foundations for direct regulatory action of the local TH-immunoreactive neurons on parasympathetic efferent neurons supplying the prepyloric region of the porcine stomach. Combined injection of neuronal retrograde tracer Fast Blue into the stomach prepyloric region with TH immunostaining was used in order to visualize spatial relationship between DMX-located stomach prepyloric region supplying neuronal stomata and local TH-IR terminals. We confirmed existence of TH-immunoreactive neural terminals closely opposing the stomach prepyloric region innervating neurons at the porcine DMX area. The observed spatial relationship points out the possibility of indirect catecholaminergic control of the stomach function exerted through preganglionic parasympathetic efferent neurons in the pig.

Presynaptic precursor vesicles originate from the trans-Golgi network, promoted by the small GTPase RAB2

SummaryReliable delivery of presynaptic material, including active zone and synaptic vesicle proteins from neuronal somata to synaptic terminals is prerequisite for faithful synaptogenesis and neurotransmission. However, molecular mechanisms controlling the somatic assembly of presynaptic precursors remain insufficiently understood. Here we show that in mutants of the small GTPase RAB2 active zone and synaptic vesicle proteins accumulated in the neuronal somata at the trans-Golgi network and were consequently depleted at synaptic terminals, provoking neurotransmission deficits. The ectopic presynaptic material accumulations consisted of heterogeneous vesicles and short tubules of 40×60 nm and segregated in subfractions either positive for active zone proteins or co-positive for synaptic vesicle proteins and LAMP1, a lysosomal membrane protein. Genetically, rab2 behaved epistatic over arl8, a lysosomal adaptor controlling axonal export of precursors. Collectively, we here identified a Golgi-associated assembly sequence in presynaptic precursor vesicle biogenesis controlled by RAB2 dependent membrane remodelling and protein sorting at the trans-Golgi.

0067 An Anatomic Substrate for GABAergic Processes to Suppress Active Sleep and Promote Wakefulness in the Nucleus Pontis Oralis

Introduction Our previous electrophysiologic data have provided compelling evidence that GABAergic processes in the nucleus pontis oralis (NPO) play a critical role in the generation and maintenance of wakefulness as well as active (REM) sleep (AS). We therefore hypothesized that one of the neuronal mechanisms of GABA actions in the NPO to promote wakefulness and suppress AS is due to a direct GABAergic inhibition of NPO neurons that generate AS (AS-generator neurons). However, the anatomical substrate for this inhibition is undetermined. Accordingly, the present study was undertaken to examine whether there is any direct interaction between GABAergic neurons and glutamatergic AS-generator neurons in the NPO. Methods Adult cats were deeply anesthetized and perfused transcardially. The brainstem containing the NPO was removed, postfixed and cut into 15 μm coronal sections with a Reichert-Jung cryostat. The sections were incubated with a mixture of a rabbit polyclonal antibodies against glutamine and GABA following the procedure of double fluorescence immunohistochemistry. Results There was a large number of neuronal somata labeled by anti-glutamine antibody and terminals labeled by anti-GABA antibody in the NPO. These glutamine-positive neurons were medium to large, multipolar cells (> 20 μm), which resemble glutamatergic, AS-generator neurons that have been previously identified in the NPO. Specifically, majority of glutamatergic neuronal somata were closely apposed by multiple GABAergic terminals, indicating that AS-generator neurons in the NPO receive direct GABAergic inputs. Conclusion The present results demonstrate that a direct connection exists between glutamatergic AS-generator neurons and GABAergic processes in the NPO. These data provide the anatomical evidence which supports our hypothesis that the pontine GABAergic control of wakefulness and active sleep is partially mediated via GABAergic processes project to NPO AS-generator neurons that suppress the activity of these cells. Support NS092383

Toxoplasma induces stripping of perisomatic inhibitory synapses

ABSTRACTInfection and inflammation within the brain induces changes in neuronal connectivity and function. The intracellular protozoan parasite, Toxoplasma gondii, is one pathogen that infects the brain and can cause encephalitis and seizures. Persistent infection by this parasite is also associated with behavioral alterations and an increased risk for developing psychiatric illness, including schizophrenia. Current evidence from studies in humans and mouse models suggest that both seizures and schizophrenia result from a loss or dysfunction of inhibitory synapses. In line with this, we recently reported that persistent Toxoplasma gondii infection alters the distribution of glutamic acid decarboxylase 67 (GAD67), an enzyme that catalyzes GABA synthesis in inhibitory synapses. These changes could reflect a redistribution of presynaptic machinery in inhibitory neurons or a loss of inhibitory nerve terminals. To directly assess the latter possibility, we employed serial block face scanning electron microscopy (SBFSEM) and quantified inhibitory perisomatic synapses in neocortex and hippocampus following parasitic infection. Not only did persistent infection lead to a significant loss of perisomatic synapses, it induced the ensheathment of neuronal somata by phagocytic cells. Immunohistochemical, genetic, and ultrastructural analyses revealed that these phagocytic cells included reactive microglia. Finally, ultrastructural analysis identified phagocytic cells enveloping perisomatic nerve terminals, suggesting they may participate in synaptic stripping. Thus, these results suggest that microglia contribute to perisomatic inhibitory synapse loss following parasitic infection and offer a novel mechanism as to how persistent Toxoplasma gondii infection may contribute to both seizures and psychiatric illness.MAIN POINTSToxoplasma-infection leads the loss of perisomatic inhibitory synapsesPhagocytic microglia ensheath neuronal somata following Toxoplasma-infectionMicroglia contact and envelop perisomatic nerve terminals, suggesting that Toxoplasma induces synaptic stripping

Proteoglycan (Keratan Sulfate) Barrier in Developing Human Forebrain Isolates Cortical Epileptic Networks From Deep Heterotopia, Insulates Axonal Fascicles, and Explains Why Axosomatic Synapses Are Inhibitory

Axons from deep heterotopia do not extend through U-fibers, except transmantle dysplasias. Keratan sulfate (KS) in fetal spinal cord/brainstem median septum selectively repels glutamatergic axons while enabling GABAergic commissural axons. Immunocytochemical demonstration of KS in neocortical resections and forebrain at autopsy was studied in 12 fetuses and neonates 9–41 weeks gestational age (GA), 9 infants, children, and adolescents and 5 patients with focal cortical dysplasias (FCD1a). From 9 to 15 weeks GA, no KS is seen in the cortical plate; 19-week GA reactivity is detected in the molecular zone. By 28 weeks GA, patchy granulofilamentous reactivity appears in extracellular matrix and adheres to neuronal somata with increasing intensity in deep cortex and U-fibers at term. Perifascicular KS surrounds axonal bundles of both limbs of the internal capsule and within basal ganglia from 9 weeks GA. Thalamus and globus pallidus exhibit intense astrocytic reactivity from 9 weeks GA. In FCD1a, U-fiber reactivity is normal, discontinuous or radial. Ultrastructural correlates were not demonstrated; KS is not electron-dense. Proteoglycan barrier of the U-fiber layer impedes participation of deep heterotopia in cortical epileptic networks. Perifascicular KS prevents aberrant axonal exit from or entry into long and short tracts. KS adhesion to neuronal somatic membranes may explain inhibitory axosomatic synapses.

Perineuronal Nets in the Superior Olivary Complex

This chapter addresses perineuronal nets in the superior olivary complex, a collection of nuclei in the auditory brainstem that are involved in the processing of sound source location. Perineuronal nets, a specific form of extracellular matrix, are believed to control synaptic plasticity. They surround neuronal somata and dendrites of specific types of neurons, among which are many neurons of the superior olivary complex. The chapter describes the distribution of perineuronal nets in the superior olivary complex, focusing on controversial results and discussing underlying reasons. In addition, it considers the development of perineuronal nets and highlights differences between the main components of perineuronal nets, including the proteoglycans aggrecan, brevican, and neurocan. Finally, it introduces current concepts on the function of perineuronal nets that are specifically based on experimental data collected in the superior olivary complex and point to a contribution of perineuronal nets to synaptic transmission and neuronal excitability.