Acute total sleep deprivation (SD) impairs memory consolidation, attention, working memory and perception. Structural, electrophysiological and molecular experimental approaches provided evidences for the involvement of sleep in synaptic functions. Despite the wide scientific interest on the effects of sleep on the synapse, there is a lack of systematic investigation of sleep-related changes in the synaptic proteome. We isolated parietal cortical and thalamic synaptosomes of rats after 8h of total SD by gentle handling and 16h after the end of deprivation to investigate the short- and longer-term effects of SD on the synaptic proteome, respectively. The SD efficiency was verified by electrophysiology. Protein abundance alterations of the synaptosomes were analyzed by fluorescent two-dimensional differential gel electrophoresis and by tandem mass spectrometry. As several altered proteins were found to be involved in synaptic strength regulation, our data can support the synaptic homeostasis hypothesis function of sleep and highlight the long-term influence of SD after the recovery sleep period, mostly on cortical synapses. Furthermore, the large-scale and brain area-specific protein network change in the synapses may support both ideas of sleep-related synaptogenesis and molecular maintenance and reorganization in normal rat brain.

C1q, a member of the immune complement cascade, is implicated in the selective pruning of synapses by microglial phagocytosis. C1q-mediated synapse elimination has been shown to occur during brain development, while increased activation and complement-dependent synapse loss is observed in neurodegenerative diseases. However, the molecular mechanisms underlying C1q-controlled synaptic pruning are mostly unknown. This study addresses distortions in the synaptic proteome leading to C1q-tagged synapses. Our data demonstrated the preferential localization of C1q to the presynapse. Proteomic investigation and pathway analysis of C1q-tagged synaptosomes revealed the presence of apoptotic-like processes in C1q-tagged synapses, which was confirmed experimentally with apoptosis markers. Moreover, the induction of synaptic apoptotic-like mechanisms in a model of sensory deprivation-induced synaptic depression led to elevated C1q levels. Our results unveiled that C1q label-based synaptic pruning is triggered by and directly linked to apoptotic-like processes in the synaptic compartment.

Oxytocin is released from neurons in the paraventricular hypothalamic nucleus (PVN) in mothers upon suckling and during adult social interactions. However, neuronal pathways that activate oxytocin neurons in social contexts are not yet established. Neurons in the posterior intralaminar complex of the thalamus (PIL), which contain tuberoinfundibular peptide 39 (TIP39) and are activated by pup exposure in lactating mothers, provide a candidate projection. Innervation of oxytocin neurons by TIP39 neurons was examined by double labeling in combination with electron microscopy and retrograde tract-tracing. Potential classic neurotransmitters in TIP39 neurons were investigated by in situ hybridization histochemistry. Neurons activated after encounter with a familiar conspecific female in a familiar environment were mapped with the c-Fos technique. PVN and the supraoptic nucleus oxytocin neurons were closely apposed by an average of 2.0 and 0.4 TIP39 terminals, respectively. Asymmetric (presumed excitatory) synapses were found between TIP39 terminals and cell bodies of oxytocin neurons. In lactating rats, PIL TIP39 neurons were retrogradely labeled from the PVN. TIP39 neurons expressed vesicular glutamate transporter 2 but not glutamic acid decarboxylase 67. PIL contained a markedly increased number of c-Fos-positive neurons in response to social encounter with a familiar conspecific female. Furthermore, the PIL received ascending input from the spinal cord and the inferior colliculus. Thus, TIP39 neurons in the PIL may receive sensory input in response to social interactions and project to the PVN to innervate and excite oxytocin neurons, suggesting that the PIL-PVN projection contributes to the activation of oxytocin neurons in social contexts.

To establish synaptic proteome changes associated with motherhood, we isolated synaptosome fractions from the hypothalamus of mother rats and non-maternal control females at the 11th postpartum day. Proteomic analysis by two-dimensional differential gel electrophoresis combined with mass spectrometric protein identification established 26 significant proteins, 7 increasing and 19 decreasing protein levels in the dams. The altered proteins are mainly involved in energy homeostasis, protein folding, and metabolic processes suggesting the involvement of these cellular processes in maternal adaptations. The decrease in a significantly altered protein, complement component 1q subcomponent-binding protein (C1qbp) was validated with Western blotting. Furthermore, immunohistochemistry showed its presence in hypothalamic fibers and terminals in agreement with its presence in synaptosomes. We also found the expression of C1qbp in different hypothalamic nuclei including the preoptic area and the paraventricular hypothalamic nucleus at the protein and at the mRNA level using immunohistochemistry and in situ hybridization histochemistry, respectively. Bioinformatical network analysis revealed that cytokines, growth factors, and protein kinases are common regulators, which indicates a complex regulation of the proteome change in mothers. The results suggest that maternal responsiveness is associated with synaptic proteins level changes in the hypothalamus, and that growth factors and cytokines may govern these alterations.

CASK-interactive proteins, Caskin1 and Caskin2, are multidomain neuronal scaffold proteins. Recent data from Caskin1 knockout animals indicated only a mild role of Caskin1 in anxiety and pain perception. In this work, we show that deletion of both Caskins leads to severe deficits in novelty recognition and spatial memory. Ultrastructural analyses revealed a reduction in synaptic profiles and dendritic spine areas of CA1 hippocampal pyramidal neurons of double knockout mice. Loss of Caskin proteins impaired LTP induction in hippocampal slices, while miniature EPSCs in dissociated hippocampal cultures appeared to be unaffected. In cultured Caskin knockout hippocampal neurons, overexpressed Caskin1 was enriched in dendritic spine heads and increased the amount of mushroom-shaped dendritic spines. Chemically induced LTP (cLTP) mediated enlargement of spine heads was augmented in the knockout mice and was not influenced by Caskin1. Immunocytochemistry and immunoprecipitation confirmed that Shank2, a master scaffold of the postsynaptic density, and Caskin1 co-localized within the same complex. Phosphorylation of AMPA receptors was specifically altered by Caskin deficiency and was not elevated by cLTP treatment further. Taken together, our results prove a previously unnoticed postsynaptic role of Caskin scaffold proteins and indicate that Caskins influence learning abilities via regulating spine morphology and AMPA receptor localisation.

GABAergic interneurons in the hippocampus provide for local and long-distance coordination of neurons in functionally connected areas. Vasoactive intestinal peptide-expressing (VIP+) interneurons occupy a distinct niche in circuitry as many of them specialize in innervating GABAergic cells, thus providing network disinhibition. In the CA1 hippocampus, VIP+ interneuron-selective cells target local interneurons. Here, we discover a type of VIP+ neuron whose axon innervates CA1 and also projects to the subiculum (VIP-LRPs). VIP-LRPs show specific molecular properties and target interneurons within the CA1 area but both interneurons and pyramidal cells within subiculum. They are interconnected through gap junctions but demonstrate sparse spike coupling in vitro. In awake mice, VIP-LRPs decrease their activity during theta-run epochs and are more active during quiet wakefulness but not coupled to sharp-wave ripples. Together, the data provide evidence for VIP interneuron molecular diversity and functional specialization in controlling cell ensembles along the hippocampo-subicular axis.

During autophagy, double-membrane autophagosomes deliver sequestered cytoplasmic content to late endosomes and lysosomes for degradation. The molecular mechanism of autophagosome maturation is still poorly characterized. The small GTPase Rab11 regulates endosomal traffic and is thought to function at the level of recycling endosomes. We show that loss of Rab11 leads to accumulation of autophagosomes and late endosomes in Drosophila melanogaster. Rab11 translocates from recycling endosomes to autophagosomes in response to autophagy induction and physically interacts with Hook, a negative regulator of endosome maturation. Hook anchors endosomes to microtubules, and we show that Rab11 facilitates the fusion of endosomes and autophagosomes by removing Hook from mature late endosomes and inhibiting its homodimerization. Thus induction of autophagy appears to promote autophagic flux by increased convergence with the endosomal pathway.

Lipid droplets (LDs) are common organelles of the majority of eukaryotic cell types. Their biological significance has been extensively studied in mammalian liver cells and white adipose tissue. Although the central nervous system contains the highest relative amount and the largest number of different lipid species, neither the spatial nor the temporal distribution of LDs has been described. In this study, we used the brain of the fruitfly, Drosophila melanogaster, to investigate the neuroanatomy of LDs. We demonstrated that LDs are exclusively localised in glial cells but not in neurons in the larval nervous system. We showed that the brain's LD pool, rather than being constant, changes dynamically during development and reaches its highest value at the beginning of metamorphosis. LDs are particularly enriched in cortex glial cells located close to the brain surface. These specialized superficial cortex glial cells contain the highest amount of LDs among glial cell types and encapsulate neuroblasts and their daughter cells. Superficial cortex glial cells, combined with subperineurial glial cells, express the Drosophila fatty acid binding protein (Dfabp), as we have demonstrated through light- and electron microscopic immunocytochemistry. To the best of our best knowledge this is the first study that describes LD neuroanatomy in the Drosophila larval brain.

The synapse is a particularly important compartment of neurons. To reveal its molecular characteristics we isolated whole brain synaptic (sMito) and non-synaptic mitochondria (nsMito) from the mouse brain with purity validated by electron microscopy and fluorescence activated cell analysis and sorting. Two-dimensional differential gel electrophoresis and mass spectrometry based proteomics revealed 22 proteins with significantly higher and 34 proteins with significantly lower levels in sMito compared to nsMito. Expression differences in some oxidative stress related proteins, such as superoxide dismutase [Mn] (Sod2) and complement component 1Q subcomponent-binding protein (C1qbp), as well as some tricarboxylic acid cycle proteins, including isocitrate dehydrogenase subunit alpha (Idh3a) and ATP-forming β subunit of succinyl-CoA ligase (SuclA2), were verified by Western blot, the latter two also by immunohistochemistry. The data suggest altered tricarboxylic acid metabolism in energy supply of synapse while the marked differences in Sod2 and C1qbp support high sensitivity of synapses to oxidative stress. Further functional clustering demonstrated that proteins with higher synaptic levels are involved in synaptic transmission, lactate and glutathione metabolism. In contrast, mitochondrial proteins associated with glucose, lipid, ketone metabolism, signal transduction, morphogenesis, protein synthesis and transcription were enriched in nsMito. Altogether, the results suggest a specifically tuned composition of synaptic mitochondria.

Atg6 (Beclin 1 in mammals) is a core component of the Vps34 PI3K (III) complex, which promotes multiple vesicle trafficking pathways. Atg6 and Vps34 form two distinct PI3K (III) complexes in yeast and mammalian cells, either with Atg14 or with UVRAG. The functions of these two complexes are not entirely clear, as both Atg14 and UVRAG have been suggested to regulate both endocytosis and autophagy. In this study, we performed a microscopic analysis of UVRAG, Atg14, or Atg6 loss-of-function cells in the developing Drosophila wing. Both autophagy and endocytosis are seriously impaired and defective endolysosomes accumulate upon loss of Atg6. We show that Atg6 is required for the downregulation of Notch and Wingless signaling pathways; thus it is essential for normal wing development. Moreover, the loss of Atg6 impairs cell polarity. Atg14 depletion results in autophagy defects with no effect on endocytosis or cell polarity, while the silencing of UVRAG phenocopies all but the autophagy defect of Atg6 depleted cells. Thus, our results indicate that the UVRAG-containing PI3K (III) complex is required for receptor downregulation through endolysosomal degradation and for the establishment of proper cell polarity in the developing wing, while the Atg14-containing complex is involved in autophagosome formation.

An increasing number of studies have revealed associations between pre- and perinatal immune activation and the development of schizophrenia and autism spectrum disorders (ASDs). Accordingly, neuroimmune crosstalk has a considerably large impact on brain development during early ontogenesis. While a plethora of heterogeneous abnormalities have already been described in established maternal immune activation (MIA) rodent and primate animal models, which highly correlate to those found in human diseases, the underlying molecular background remains obscure. In the current study, we describe the long-term effects of MIA on the neocortical pre- and postsynaptic proteome of adolescent rat offspring in detail. Molecular differences were revealed in sub-synaptic fractions, which were first thoroughly characterized using independent methods. The widespread proteomic examination of cortical samples from offspring exposed to maternal lipopolysaccharide administration at embryonic day 13.5 was conducted via combinations of different gel-based proteomic techniques and tandem mass spectrometry. Our experimentally validated proteomic data revealed more pre- than postsynaptic protein level changes in the offspring. The results propose the relevance of altered synaptic vesicle recycling, cytoskeletal structure and energy metabolism in the presynaptic region in addition to alterations in vesicle trafficking, the cytoskeleton and signal transduction in the postsynaptic compartment in MIA offspring. Differing levels of the prominent signaling regulator molecule calcium/calmodulin-dependent protein kinase II in the postsynapse was validated and identified specifically in the prefrontal cortex. Finally, several potential common molecular regulators of these altered proteins, which are already known to be implicated in schizophrenia and ASD, were identified and assessed. In summary, unexpectedly widespread changes in the synaptic molecular machinery in MIA rats were demonstrated which might underlie the pathological cortical functions that are characteristic of schizophrenia and ASD.

Elucidating the molecular mechanisms underlying the functional diversity of synapses requires a high-resolution, sensitive, diffusion-free, quantitative localization method that allows the determination of many proteins in functionally characterized individual synapses. Array tomography permits the quantitative analysis of single synapses but has limited sensitivity, and its application to functionally characterized synapses is challenging. Here, we aim to overcome these limitations by searching the parameter space of different fixation, resin, embedding, etching, retrieval, and elution conditions. Our optimizations reveal that etching epoxy-resin-embedded ultrathin sections with Na-ethanolate and treating them with SDS dramatically increase the labeling efficiency of synaptic proteins. We also demonstrate that this method is ideal for the molecular characterization of individual synapses following paired recordings, two-photon [Ca2+] or glutamate-sensor (iGluSnFR) imaging. This method fills a missing gap in the toolbox of molecular and cellular neuroscience, helping us to reveal how molecular heterogeneity leads to diversity in function.