Aberrant hippocampal Ca2+ micro-waves following synapsin-dependent adeno-associated viral expression of Ca2+ indicators.

Genetically encoded calcium indicators (GECIs) such as GCaMP are invaluable tools in neuroscience to monitor neuronal activity using optical imaging. The viral transduction of GECIs is commonly used to target expression to specific brain regions, can be conveniently used with any mouse strain of interest without the need for prior crossing with a GECI mouse line and avoids potential hazards due to the chronic expression of GECIs during development. A key requirement for monitoring neuronal activity with an indicator is that the indicator itself minimally affects activity. Here, using common adeno-associated viral (AAV) transduction procedures, we describe spatially confined aberrant Ca2+ micro-waves slowly travelling through the hippocampus following expression of GCaMP6, GCaMP7 or R-CaMP1.07 driven by the synapsin promoter with AAV-dependent gene transfer, in a titre-dependent fashion. Ca2+ micro-waves developed in hippocampal CA1 and CA3, but not dentate gyrus (DG) nor neocortex, were typically first observed at 4 weeks after viral transduction, and persisted up to at least 8 weeks. The phenomenon was robust, observed across laboratories with various experimenters and setups. Our results indicate that aberrant hippocampal Ca2+ micro-waves depend on the promoter and viral titre of the GECI, density of expression as well as the targeted brain region. We used an alternative viral transduction method of GCaMP which avoids this artifact. The results show that commonly used Ca2+-indicator AAV transduction procedures can produce artefactual Ca2+ responses. Our aim is to raise awareness in the field of these artefactual transduction-induced Ca2+ micro-waves and we provide a potential solution.

Serum Metabolome Analysis Identified Amino-Acid Metabolism Associated With Pain in People With Symptomatic Knee Osteoarthritis - A Cross-Sectional Study.

Osteoarthritis (OA) is the most common arthritis affecting synovial joints such as knees and hips of millions of people globally. Usage-related joint pain and reduced function are the most common symptoms experienced by people with OA. To improve pain management, there is a need to identify validated biomarkers predicting therapeutic responses in targeted clinical trials. Our study aimed to identify the metabolic biomarkers for pain and pressure pain detection thresholds (PPTs) in participants with knee pain and symptomatic OA using metabolic phenotyping. Metabolite and cytokine measurements were done on serum samples using LC-MS/MS (liquid gas chromatography integrated magnetic resonance mass spectrometry) and Human Proinflammatory panel 1 kit respectively. Regression analysis was done in a test (n = 75) and replication study (n = 79) to investigate the metabolites associated with current knee pain scores and pressure pain detection thresholds (PPTs). Meta-analysis and correlation were done estimating precision of associated metabolites and identifying relationship between significant metabolites and cytokines respectively. Acyl ornithine, carnosine, cortisol, cortisone, cystine, DOPA, glycolithocholic acid sulphate (GLCAS), phenylethylamine (PEA) and succinic acid were found to be significantly (FDR <.1) associated with pain scores in meta-analysis of both studies. IL-10, IL-13, IL-1ß, IL2, IL8 and TNF-α were also found to be associated with the significant metabolites. Significant associations of these metabolites and inflammatory markers with knee pain suggests that targeting relevant pathways of amino acid and cholesterol metabolism may modulate cytokines and these could be targeted as novel therapeutics development to improve knee pain and OA management. PERSPECTIVE: Foreseeing the global burden of knee pain in Osteoarthritis (OA) and adverse effects of current pharmacological therapies, this study is envisaged to investigate serum metabolites and molecular pathways involved in knee pain. The replicated metabolites in this study suggests targeting amino-acid pathways for better management of OA knee pain.

Ste20-like Kinase Is Critical for Inhibitory Synapse Maintenance and Its Deficiency Confers a Developmental Dendritopathy.

The size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that, during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ third-order dendrites both in male and in female mice. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, whereas excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and inhibitory synapse maintenance.SIGNIFICANCE STATEMENT We show that dysmorphic neurons of human epileptogenic brain lesions have decreased levels of the Ste20-like kinase (SLK). Decreasing SLK expression in mouse neurons revealed that SLK has essential functions in forming the neuronal dendritic tree and in maintaining inhibitory connections with neighboring neurons.

Regulation of Cl--HCO3- exchangers by cAMP-dependent protein kinase in adult rat hippocampal CA1 neurons.

The contributions of HCO(3)(-)-dependent, DIDS-sensitive mechanisms to the maintenance of steady-state pH(i), and the regulation of their activities by cAMP-dependent protein kinase (PKA), were investigated in CA1 neurons with the H(+)-sensitive fluorophore, BCECF. The addition of HCO(3)(-)/CO(2) to neurons with "low" (pH(i) < or = 7.20) and "high" (pH(i) > 7.20) initial pH(i) values under Hepes-buffered conditions, increased and decreased steady-state pH(i), respectively. Conversely, under HCO(3)(-)/CO(2)-buffered conditions, DIDS caused pH(i) to decrease and increase in neurons with low and high initial pH(i) values, respectively. In the presence, but not the absence, of HCO(3)(-), the PKA inhibitor Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS; 50 microM) evoked DIDS-sensitive increases and decreases in pH(i) in neurons with low and high initial pH(i) values, respectively. In contrast, in neurons with low initial pH(i) values, activation of PKA with the Sp isomer of cAMPS (Sp-cAMPS; 25 microM) elicited increases in pH(i) that were smaller in the presence than in the absence of HCO(3)(-), whereas in neurons with high initial pH(i) values, Sp-cAMPS-evoked rises in pH(i) were larger in the presence than in the absence of HCO(3)(-); the differences between the effects of Sp-cAMPS on pH(i) under the different buffering conditions were attenuated by DIDS. Consistent with the possibility that changes in the activities of HCO(3)(-)-dependent, DIDS-sensitive mechanisms contribute to the steady-state pH(i) changes evoked by the PKA modulators, in neurons with initial pH(i) values < or = 7.20, Rp-cAMPS concurrently inhibited Na(+)-independent Cl(-)-HCO(3)(-) exchange and stimulated Na(+)-dependent Cl(-)-HCO(3)(-) exchange; in contrast, Sp-cAMPS concurrently stimulated Na(+)-independent Cl(-)-HCO(3)(-) exchange and inhibited Na(+)-dependent Cl(-)-HCO(3)(-) exchange. Data from a limited number of neurons with initial pH(i) values > 7.20 suggested that the directions of the reciprocal changes in anion exchange activities (inhibition or stimulation) evoked by Rp- and Sp-cAMPS may be opposite in cells with low vs. high resting pH(i) values. Taken together, the results indicate that the effects of modulating PKA activity on steady-state pH(i) in rat CA1 neurons under HCO(3)(-)/CO(2)-buffered conditions reflect not only changes in Na(+)-H(+) exchange activity but also changes in Na(+)-dependent and Na(+)-independent Cl(-)-HCO(3)(-) exchange activity that, in turn, may be dependent upon the initial pH(i).