Mass spectrometry is the driving force behind current brain proteome analysis. In a typical proteomics approach, a protein isolate is digested into tryptic peptides and then analyzed by liquid ...chromatography-mass spectrometry. The recent advancements in data independent acquisition (DIA) mass spectrometry provide higher sensitivity and protein coverage than the classic data dependent acquisition. DIA cycles through a pre-defined set of peptide precursor isolation windows stepping through 400-1,200 m/z across the whole liquid chromatography gradient. All peptides within an isolation window are fragmented simultaneously and detected by tandem mass spectrometry. Peptides are identified by matching the ion peaks in a mass spectrum to a spectral library that contains information of the peptide fragment ions' pattern and its chromatography elution time. Currently, there are several reports on DIA in brain research, in particular the quantitative analysis of cellular and synaptic proteomes to reveal the spatial and/or temporal changes of proteins that underlie neuronal plasticity and disease mechanisms. Protocols in DIA are continuously improving in both acquisition and data analysis. The depth of analysis is currently approaching proteome-wide coverage, while maintaining high reproducibility in a stable and standardisable MS environment. DIA can be positioned as the method of choice for routine proteome analysis in basic brain research and clinical applications.
Long-term depression (LTD) of synaptic strength can take multiple forms and contribute to circuit remodeling, memory encoding or erasure. The generic term LTD encompasses various induction pathways, ...including activation of NMDA, mGlu or P2X receptors. However, the associated specific molecular mechanisms and effects on synaptic physiology are still unclear. We here compare how NMDAR- or P2XR-dependent LTD affect synaptic nanoscale organization and function in rodents. While both LTDs are associated with a loss and reorganization of synaptic AMPARs, only NMDAR-dependent LTD induction triggers a profound reorganization of PSD-95. This modification, which requires the autophagy machinery to remove the T19-phosphorylated form of PSD-95 from synapses, leads to an increase in AMPAR surface mobility. We demonstrate that these post-synaptic changes that occur specifically during NMDAR-dependent LTD result in an increased short-term plasticity improving neuronal responsiveness of depressed synapses. Our results establish that P2XR- and NMDAR-mediated LTD are associated to functionally distinct forms of LTD.
Generation of neuronal cultures from induced pluripotent stem cells (hiPSCs) serve the studies of human brain disorders. However we lack neuronal networks with balanced excitatory-inhibitory ...activities, which are suitable for single cell analysis. We generated low-density networks of hPSC-derived GABAergic and glutamatergic cortical neurons. We used two different co-culture models with astrocytes. We show that these cultures have balanced excitatory-inhibitory synaptic identities using confocal microscopy, electrophysiological recordings, calcium imaging and mRNA analysis. These simple and robust protocols offer the opportunity for single-cell to multi-level analysis of patient hiPSC-derived cortical excitatory-inhibitory networks; thereby creating advanced tools to study disease mechanisms underlying neurodevelopmental disorders.
Functional genetic analyses in mice rely on efficient and in-depth characterization of the behavioral spectrum. Automated home-cage observation can provide a systematic and efficient screening method ...to detect unexplored, novel behavioral phenotypes. Here, we analyzed high-throughput automated home-cage data using existing and novel concepts, to detect a plethora of genetic differences in spontaneous behavior in a panel of commonly used inbred strains (129S1/SvImJ, A/J, C3H/HeJ, C57BL/6J, BALB/cJ, DBA/2J, NOD/LtJ, FVB/NJ, WSB/EiJ, PWK/PhJ and CAST/EiJ). Continuous video-tracking observations of sheltering behavior and locomotor activity were segmented into distinguishable behavioral elements, and studied at different time scales, yielding a set of 115 behavioral parameters of which 105 showed highly significant strain differences. This set of 115 parameters was highly dimensional; principal component analysis identified 26 orthogonal components with eigenvalues above one. Especially novel parameters of sheltering behavior and parameters describing aspects of motion of the mouse in the home-cage showed high genetic effect sizes. Multi-day habituation curves and patterns of behavior surrounding dark/light phase transitions showed striking strain differences, albeit with lower genetic effect sizes. This spontaneous home-cage behavior study demonstrates high dimensionality, with a strong genetic contribution to specific sets of behavioral measures. Importantly, spontaneous home-cage behavior analysis detects genetic effects that cannot be studied in conventional behavioral tests, showing that the inclusion of a few days of undisturbed, labor extensive home-cage assessment may greatly aid gene function analyses and drug target discovery.
Encoding and retrieval of contextual memories is initially mediated by sparsely activated neurons, so-called engram cells, in the hippocampus. Subsequent memory persistence is thought to depend on ...network-wide changes involving progressive contribution of cortical regions, a process referred to as systems consolidation. Using a viral-based TRAP (targeted recombination in activated populations) approach, we studied whether consolidation of contextual fear memory by neurons in the medial prefrontal cortex (mPFC) is modulated by memory strength and CREB function. We demonstrate that activity of a small subset of mPFC neurons is sufficient and necessary for remote memory expression, but their involvement depends on the strength of conditioning. Furthermore, selective disruption of CREB function in mPFC engram cells after mild conditioning impairs remote memory expression. Together, our data demonstrate that memory consolidation by mPFC engram cells requires CREB-mediated transcription, with the functionality of this network hub being gated by memory strength.
More than one-third of all people are estimated to experience mild to severe cognitive impairment as they age. Acetylcholine (ACh) levels in the brain diminish with aging, and nicotinic ACh receptor ...(nAChR) stimulation is known to enhance cognitive performance. The prefrontal cortex (PFC) is involved in a range of cognitive functions and is thought to mediate attentional focus. We found that mice carrying nAChR β2-subunit deletions have impaired attention performance. Efficient lentiviral vector—mediated reexpression of functional β2-subunit—containing nAChRs in PFC neurons of the prelimbic area (PrL) completely restored the attentional deficit but did not affect impulsive and motivational behavior. Our findings show that β2-subunit expression in the PrL PFC is sufficient for endogenous nAChR-mediated cholinergic regulation of attentional performance.
Copy-number variants of the CYFIP1 gene in humans have been linked to autism spectrum disorders (ASD) and schizophrenia (SCZ), two neuropsychiatric disorders characterized by defects in brain ...connectivity. Here, we show that CYFIP1 plays an important role in brain functional connectivity and callosal functions. We find that Cyfip1-heterozygous mice have reduced functional connectivity and defects in white matter architecture, similar to phenotypes found in patients with ASD, SCZ and other neuropsychiatric disorders. Cyfip1-deficient mice also present decreased myelination in the callosal axons, altered presynaptic function, and impaired bilateral connectivity. Finally, Cyfip1 deficiency leads to abnormalities in motor coordination, sensorimotor gating and sensory perception, which are also known neuropsychiatric disorder-related symptoms. These results show that Cyfip1 haploinsufficiency compromises brain connectivity and function, which might explain its genetic association to neuropsychiatric disorders.
Schizophrenia is a highly polygenic brain disorder. The main hypothesis for disease etiology in schizophrenia primarily focuses on the role of dysfunctional synaptic transmission. Previous studies ...have therefore directed their investigations toward the role of neuronal dysfunction. However, recent studies have shown that apart from neurons, glial cells also play a major role in synaptic transmission. Therefore, we investigated the potential causal involvement of the 3 principle glial cell lineages in risk to schizophrenia. We performed a functional gene set analysis to test for the combined effects of genetic variants in glial type-specific genes for association with schizophrenia. We used genome-wide association data from the largest schizophrenia sample to date, including 13 689 cases and 18 226 healthy controls. Our results show that astrocyte and oligodendrocyte gene sets, but not microglia gene sets, are associated with an increased risk for schizophrenia. The astrocyte and oligodendrocyte findings are related to astrocyte signaling at the synapse, myelin membrane integrity, glial development, and epigenetic control. Together, these results show that genetic alterations underlying specific glial cell type functions increase susceptibility to schizophrenia and provide evidence that the neuronal hypothesis of schizophrenia should be extended to include the role of glia.
Many psychiatric disorders emerge during adolescence. The study of executive functions in animal models of these disorders critically requires short-duration tasks measuring these functions before ...the animal ages. Here, a novel 5-choice serial reaction time task (5-CSRTT) protocol is presented, to measure attention and impulsivity within one week, without scheduled food deprivation and with little animal handling. Mice were allowed 24-h/day task access from their home-cage, during which they could self-pace task progression and earn unlimited food rewards depending on task performance. Manipulation of task parameters in this self-paced 5-CSRTT protocol (SP-5C) affected attentional performance and impulsivity to a similar extent as previously observed in the 5-CSRTT. Task activity followed intrinsic circadian rhythm, distinctive for the SP-5C protocol, with task performance stable over the day. The sensitivity of the SP-5C protocol to detect strain differences between C57BL/6J, DBA/2 J, BXD16 and BXD62 mice was demonstrated as well as its suitability for testing adolescent mice. Acute administration of the muscarinic acetylcholine receptor antagonist scopolamine impaired attentional performance, providing initial pharmacological validation of the task. The SP-5C substantially shortens the assessment of impulsivity and attention, increases test efficiency and enables the assessment of adolescent mouse models of psychiatric disorders.
CKAMP44, identified here by a proteomic approach, is a brain-specific type I transmembrane protein that associates with AMPA receptors in synaptic spines. CKAMP44 expressed in Xenopus oocytes reduced ...GluA1- and A2-mediated steady-state currents, but did not affect kainate- or N-methyl- D-aspartate (NMDA) receptor-mediated currents. Mouse hippocampal CA1 pyramidal neurons expressed CKAMP44 at low abundance, and overexpression of CKAMP44 led to stronger and faster AMPA receptor desensitization, slower recovery from desensitization, and a reduction in the paired-pulse ratio of AMPA currents. By contrast, dentate gyrus granule cells exhibited strong CKAMP44 expression, and CKAMP44 knockout increased the paired-pulse ratio of AMPA currents in lateral and medial perforant path-granule cell synapses. CKAMP44 thus modulates short-term plasticity at specific excitatory synapses.
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