Memory encoding and retrieval by retrosplenial parvalbumin interneurons are impaired in Alzheimer's disease model mice.

Memory deficits in Alzheimer's disease (AD) show a strong link with GABAergic interneuron dysfunctions.1,2,3,4,5,6,7 The ensemble dynamics of GABAergic interneurons represent memory encoding and retrieval,8,9,10,11,12 but how GABAergic interneuron dysfunction affects inhibitory ensemble dynamics in AD is unknown. As the retrosplenial cortex (RSC) is critical for episodic memory13,14,15,16 and is affected by ß-amyloid accumulation in early AD,17,18,19,20,21 we address this question by performing Ca2+ imaging in RSC parvalbumin (PV)-expressing interneurons during a contextual fear memory task in healthy control mice and the 5XFAD mouse model of AD. We found that populations of PV interneurons responsive to aversive electric foot shocks during contextual fear conditioning (shock-responsive) significantly decreased in the 5XFAD mice, indicating dysfunctions in the recruitment of memory-encoding PV interneurons. In the control mice, ensemble activities of shock-responsive PV interneurons were selectively upregulated during the freezing epoch of the contextual fear memory retrieval, manifested by synaptic potentiation of PV interneuron-mediated inhibition. However, such changes in ensemble dynamics during memory retrieval and synaptic plasticity were both absent in the 5XFAD mice. Optogenetic silencing of PV interneurons during contextual fear conditioning in the control mice mimicked the memory deficits in the 5XFAD mice, while optogenetic activation of PV interneurons in the 5XFAD mice restored memory retrieval. These results demonstrate the critical roles of contextual fear memory-encoding PV interneurons for memory retrieval. Furthermore, synaptic dysfunction of PV interneurons may disrupt the recruitment of PV interneurons and their ensemble dynamics underlying contextual fear memory retrieval, subsequently leading to memory deficits in AD.

Molecular profiling and specific targeting of gemcitabine-resistant subclones in heterogeneous pancreatic cancer cell populations.

Purpose: Chemotherapy is pivotal in the multimodal treatment of pancreatic ductal adenocarcinoma (PDAC). Technical advances unveiled a high degree of inter- and intratumoral heterogeneity. We hypothesized that intratumoral heterogeneity (ITH) impacts response to gemcitabine treatment and demands specific targeting of resistant subclones. Methods: Using single cell-derived cell lines (SCDCLs) from the classical cell line BxPC3 and the basal-like cell line Panc-1, we addressed the effect of ITH on response to gemcitabine treatment. Results: Individual SCDCLs of both parental tumor cell populations showed considerable heterogeneity in response to gemcitabine. Unsupervised PCA including the 1,000 most variably expressed genes showed a clustering of the SCDCLs according to their respective sensitivity to gemcitabine treatment for BxPC3, while this was less clear for Panc-1. In BxPC3 SCDCLs, enriched signaling pathways EMT, TNF signaling via NfKB, and IL2STAT5 signaling correlated with more resistant behavior to gemcitabine. In Panc-1 SCDCLs MYC targets V1 and V2 as well as E2F targets were associated with stronger resistance. We used recursive feature elimination for Feature Selection in order to compute sets of proteins that showed strong association with the response to gemcitabine. The optimal protein set calculated for Panc-1 comprised fewer proteins in comparison to the protein set determined for BxPC3. Based on molecular profiles, we could show that the gemcitabine-resistant SCDCLs of both BxPC3 and Panc-1 are more sensitive to the BET inhibitor JQ1 compared to the respective gemcitabine-sensitive SCDCLs. Conclusion: Our model system of SCDCLs identified gemcitabine-resistant subclones and provides evidence for the critical role of ITH for treatment response in PDAC. We exploited molecular differences as the basis for differential response and used these for more targeted therapy of resistant subclones.

Contribution of agricultural land conversion to global GHG emissions: A meta-analysis.

Greenhouse gases (GHG) have extensive environmental effects by trapping heat and causing climate change and air pollution. Land plays a key role in the global cycles of GHG (i.e., carbon dioxide (CO2), methane (CH4), and nitrogen oxide (N2O)), and land use change (LUC) can lead to the release of such gases into the atmosphere or the removal of them from the atmosphere. One of the most common forms of LUC is agricultural land conversion (ALC) where agricultural lands are converted for other uses. This study aimed to review 51 original papers from 1990 to 2020 that investigate the contribution of ALC to GHG emissions from a spatiotemporal perspective using a meta-analysis method. The results of spatiotemporal effects on GHG emissions showed that the effects were significant. The emissions were affected by different continent regions representing the spatial effects. The most significant spatial effect was relevant to African and Asian countries. In addition, the quadratic relationship between ALC and GHG emissions had the highest significant coefficients, showing an upward concave curve. Therefore, increasing ALC to more than 8 % of available land led to increasing GHG emissions during the economic development process. The implications of the current study are important for policymakers from two perspectives. First, to achieve sustainable economic development, policymaking should prevent the conversion of more than 90 % of agricultural land to other uses based on the turning point of the second model. Second, policies to control global GHG emissions should take into account spatial effects (e.g., continental Africa and Asia), which show the highest contribution to GHG emissions.

Panomics reveals patient individuality as the major driver of colorectal cancer progression.

BACKGROUND: Colorectal cancer (CRC) is one of the most prevalent cancers, with over one million new cases per year. Overall, prognosis of CRC largely depends on the disease stage and metastatic status. As precision oncology for patients with CRC continues to improve, this study aimed to integrate genomic, transcriptomic, and proteomic analyses to identify significant differences in expression during CRC progression using a unique set of paired patient samples while considering tumour heterogeneity. METHODS: We analysed fresh-frozen tissue samples prepared under strict cryogenic conditions of matched healthy colon mucosa, colorectal carcinoma, and liver metastasis from the same patients. Somatic mutations of known cancer-related genes were analysed using Illumina's TruSeq Amplicon Cancer Panel; the transcriptome was assessed comprehensively using Clariom D microarrays. The global proteome was evaluated by liquid chromatography-coupled mass spectrometry (LC‒MS/MS) and validated by two-dimensional difference in-gel electrophoresis. Subsequent unsupervised principal component clustering, statistical comparisons, and gene set enrichment analyses were calculated based on differential expression results. RESULTS: Although panomics revealed low RNA and protein expression of CA1, CLCA1, MATN2, AHCYL2, and FCGBP in malignant tissues compared to healthy colon mucosa, no differentially expressed RNA or protein targets were detected between tumour and metastatic tissues. Subsequent intra-patient comparisons revealed highly specific expression differences (e.g., SRSF3, OLFM4, and CEACAM5) associated with patient-specific transcriptomes and proteomes. CONCLUSION: Our research results highlight the importance of inter- and intra-tumour heterogeneity as well as individual, patient-paired evaluations for clinical studies. In addition to changes among groups reflecting CRC progression, we identified significant expression differences between normal colon mucosa, primary tumour, and liver metastasis samples from individuals, which might accelerate implementation of precision oncology in the future.

Transcriptome and Proteome Analysis in LUHMES Cells Overexpressing Alpha-Synuclein.

LUHMES cells share many characteristics with human dopaminergic neurons in the substantia nigra, the cells, the demise of which is responsible for the motor symptoms in Parkinson's disease (PD). LUHMES cells can, therefore, be used bona fide as a model to study pathophysiological processes involved in PD. Previously, we showed that LUHMES cells degenerate after 6 days upon overexpression of wild-type alpha-synuclein. In the present study, we performed a transcriptome and proteome expression analysis in alpha-synuclein-overexpressing cells and GFP-expressing control cells in order to identify genes and proteins that are differentially regulated upon overexpression of alpha-synuclein. The analysis was performed 4 days after the initiation of alpha-synuclein or GFP overexpression, before the cells died, in order to identify processes that preceded cell death. After adjustments for multiple testing, we found 765 genes being differentially regulated (439 upregulated, 326 downregulated) and 122 proteins being differentially expressed (75 upregulated, 47 downregulated). In total, 21 genes and corresponding proteins were significantly differentially regulated in the same direction in both datasets, of these 13 were upregulated and 8 were downregulated. In total, 13 genes and 9 proteins were differentially regulated in our cell model, which had been previously associated with PD in recent genome-wide association studies (GWAS). In the gene ontology (GO) analysis of all upregulated genes, the top terms were "regulation of cell death," "positive regulation of programmed cell death," and "regulation of apoptotic signaling pathway," showing a regulation of cell death-associated genes and proteins already 2 days before the cells started to die. In the GO analysis of the regulated proteins, among the strongest enriched GO terms were "vesicle," "synapse," and "lysosome." In total, 33 differentially regulated proteins were associated with synapses, and 12 differentially regulated proteins were associated with the "lysosome", suggesting that these intracellular mechanisms, which had been previously associated with PD, also play an important role in our cell model.

Protein Expression of AEBP1, MCM4, and FABP4 Differentiate Osteogenic, Adipogenic, and Mesenchymal Stromal Stem Cells.

Mesenchymal stem cells (MSCs) gain an increasing focus in the field of regenerative medicine due to their differentiation abilities into chondrocytes, adipocytes, and osteoblastic cells. However, it is apparent that the transformation processes are extremely complex and cause cellular heterogeneity. The study aimed to characterize differences between MSCs and cells after adipogenic (AD) or osteoblastic (OB) differentiation at the proteome level. Comparative proteomic profiling was performed using tandem mass spectrometry in data-independent acquisition mode. Proteins were quantified by deep neural networks in library-free mode and correlated to the Molecular Signature Database (MSigDB) hallmark gene set collections for functional annotation. We analyzed 4108 proteins across all samples, which revealed a distinct clustering between MSCs and cell differentiation states. Protein expression profiling identified activation of the Peroxisome proliferator-activated receptors (PPARs) signaling pathway after AD. In addition, two distinct protein marker panels could be defined for osteoblastic and adipocytic cell lineages. Hereby, overexpression of AEBP1 and MCM4 for OB as well as of FABP4 for AD was detected as the most promising molecular markers. Combination of deep neural network and machine-learning algorithms with data-independent mass spectrometry distinguish MSCs and cell lineages after adipogenic or osteoblastic differentiation. We identified specific proteins as the molecular basis for bone formation, which could be used for regenerative medicine in the future.

Open-source, Python-based, hardware and software for controlling behavioural neuroscience experiments.

Laboratory behavioural tasks are an essential research tool. As questions asked of behaviour and brain activity become more sophisticated, the ability to specify and run richly structured tasks becomes more important. An increasing focus on reproducibility also necessitates accurate communication of task logic to other researchers. To these ends, we developed pyControl, a system of open-source hardware and software for controlling behavioural experiments comprising a simple yet flexible Python-based syntax for specifying tasks as extended state machines, hardware modules for building behavioural setups, and a graphical user interface designed for efficiently running high-throughput experiments on many setups in parallel, all with extensive online documentation. These tools make it quicker, easier, and cheaper to implement rich behavioural tasks at scale. As important, pyControl facilitates communication and reproducibility of behavioural experiments through a highly readable task definition syntax and self-documenting features. Here, we outline the system's design and rationale, present validation experiments characterising system performance, and demonstrate example applications in freely moving and head-fixed mouse behaviour.

Contribution of Interneuron Subtype-Specific GABAergic Signaling to Emergent Sensory Processing in Mouse Somatosensory Whisker Barrel Cortex.

Mammalian neocortex is important for conscious processing of sensory information with balanced glutamatergic and GABAergic signaling fundamental to this function. Yet little is known about how this interaction arises despite increasing insight into early GABAergic interneuron (IN) circuits. To study this, we assessed the contribution of specific INs to the development of sensory processing in the mouse whisker barrel cortex, specifically the role of INs in early speed coding and sensory adaptation. In wild-type animals, both speed processing and adaptation were present as early as the layer 4 critical period of plasticity and showed refinement over the period leading to active whisking onset. To test the contribution of IN subtypes, we conditionally silenced action-potential-dependent GABA release in either somatostatin (SST) or vasoactive intestinal peptide (VIP) INs. These genetic manipulations influenced both spontaneous and sensory-evoked cortical activity in an age- and layer-dependent manner. Silencing SST + INs reduced early spontaneous activity and abolished facilitation in sensory adaptation observed in control pups. In contrast, VIP + IN silencing had an effect towards the onset of active whisking. Silencing either IN subtype had no effect on speed coding. Our results show that these IN subtypes contribute to early sensory processing over the first few postnatal weeks.

Neocortical inhibitory interneuron subtypes are differentially attuned to synchrony- and rate-coded information.

Neurons can carry information with both the synchrony and rate of their spikes. However, it is unknown whether distinct subtypes of neurons are more sensitive to information carried by synchrony versus rate, or vice versa. Here, we address this question using patterned optical stimulation in slices of somatosensory cortex from mouse lines labelling fast-spiking (FS) and regular-spiking (RS) interneurons. We used optical stimulation in layer 2/3 to encode a 1-bit signal using either the synchrony or rate of activity. We then examined the mutual information between this signal and the interneuron responses. We found that for a synchrony encoding, FS interneurons carried more information in the first five milliseconds, while both interneuron subtypes carried more information than excitatory neurons in later responses. For a rate encoding, we found that RS interneurons carried more information after several milliseconds. These data demonstrate that distinct interneuron subtypes in the neocortex have distinct sensitivities to synchrony versus rate codes.

Targeted Protein Quantification Using Parallel Reaction Monitoring (PRM).

Targeted proteomics represents an efficient method to quantify proteins of interest with high sensitivity and accuracy. Targeted approaches were first established for triple quadrupole instruments, but the emergence of hybrid instruments allowing for high-resolution and accurate-mass measurements of MS/MS fragment ions enabled the development of parallel reaction monitoring (PRM). In PRM analysis, specific peptides are measured as representatives of proteins in complex samples, with the full product ion spectra being acquired, allowing for identification and quantification of the peptides. Ideally, corresponding stable isotope-labeled peptides are spiked into the analyzed samples to account for technical variation and enhance the precision. Here, we describe the development of a PRM assay including the selection of appropriate peptides that fulfill the criteria to serve as unique surrogates of the targeted proteins. We depict the sequential steps of method development and the generation of calibration curves. Furthermore, we present the open-access tool CalibraCurve for the determination of the linear concentration ranges and limits of quantification (LOQ).

Comprehensive miRNome-Wide Profiling in a Neuronal Cell Model of Synucleinopathy Implies Involvement of Cell Cycle Genes.

Growing evidence suggests that epigenetic mechanisms like microRNA-mediated transcriptional regulation contribute to the pathogenesis of parkinsonism. In order to study the influence of microRNAs (miRNAs), we analyzed the miRNome 2 days prior to major cell death in α-synuclein-overexpressing Lund human mesencephalic neurons, a well-established cell model of Parkinson's disease (PD), by next-generation sequencing. The expression levels of 23 miRNAs were significantly altered in α-synuclein-overexpressing cells, 11 were down- and 12 upregulated (P < 0.01; non-adjusted). The in silico analysis of known target genes of these miRNAs was complemented by the inclusion of a transcriptome dataset (BeadChip) of the same cellular system, revealing the G0/G1 cell cycle transition to be markedly enriched. Out of 124 KEGG-annotated cell cycle genes, 15 were present in the miRNA target gene dataset and six G0/G1 cell cycle genes were found to be significantly altered upon α-synuclein overexpression, with five genes up- (CCND1, CCND2, and CDK4 at P < 0.01; E2F3, MYC at P < 0.05) and one gene downregulated (CDKN1C at P < 0.001). Additionally, several of these altered genes are targeted by miRNAs hsa-miR-34a-5p and hsa-miR-34c-5p, which also modulate α-synuclein expression levels. Functional intervention by siRNA-mediated knockdown of the cell cycle gene cyclin D1 (CCND1) confirmed that silencing of cell cycle initiation is able to substantially reduce α-synuclein-mediated cytotoxicity. The present findings suggest that α-synuclein accumulation induces microRNA-mediated aberrant cell cycle activation in post-mitotic dopaminergic neurons. Thus, the mitotic cell cycle pathway at the level of miRNAs might offer interesting novel therapeutic targets for PD.

Wood profiling by non-targeted high-resolution mass spectrometry: Part 1, Metabolite profiling in Cedrela wood for the determination of the geographical origin.

The determination of the geographical origin of wood can be highly relevant for several reasons: On the one hand, it can help to prevent illegal logging and timber trade, on the other hand, it is of special interest for archaeological artefacts made of wood, as well as for a variety of biological questions. For this reason, different extraction methods were first tested for the analysis of polar and non-polar metabolites using liquid chromatography coupled electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS). A two-phase extraction with chloroform, methanol and water proved to be particularly successful. Subsequently, cedrela (Cedrela odorata) samples from South America were measured to distinguish geographic origin. Using multivariate data analysis, numerous origin-dependent differences could be extracted. The identification of the marker substances indicated that several metabolic pathways were affected by the geographical influences, some of them probably indicating pest infections.

Competing magnetic orders in a bilayer Hubbard model with ultracold atoms.

Fermionic atoms in optical lattices have served as a useful model system in which to study and emulate the physics of strongly correlated matter. Driven by the advances of high-resolution microscopy, the current research focus is on two-dimensional systems1-3, in which several quantum phases-such as antiferromagnetic Mott insulators for repulsive interactions4-7 and charge-density waves for attractive interactions8-have been observed. However, the lattice structure of real materials, such as bilayer graphene, is composed of coupled layers and is therefore not strictly two-dimensional, which must be taken into account in simulations. Here we realize a bilayer Fermi-Hubbard model using ultracold atoms in an optical lattice, and demonstrate that the interlayer coupling controls a crossover between a planar antiferromagnetically ordered Mott insulator and a band insulator of spin-singlets along the bonds between the layers. We probe the competition of the magnetic ordering by measuring spin-spin correlations both within and between the two-dimensional layers. Our work will enable the exploration of further properties of coupled-layer Hubbard models, such as theoretically predicted superconducting pairing mechanisms9,10.

Distinct roles of parvalbumin and somatostatin interneurons in gating the synchronization of spike times in the neocortex.

Synchronization of precise spike times across multiple neurons carries information about sensory stimuli. Inhibitory interneurons are suggested to promote this synchronization, but it is unclear whether distinct interneuron subtypes provide different contributions. To test this, we examined single-unit recordings from barrel cortex in vivo and used optogenetics to determine the contribution of parvalbumin (PV)- and somatostatin (SST)-positive interneurons to the synchronization of spike times across cortical layers. We found that PV interneurons preferentially promote the synchronization of spike times when instantaneous firing rates are low (<12 Hz), whereas SST interneurons preferentially promote the synchronization of spike times when instantaneous firing rates are high (>12 Hz). Furthermore, using a computational model, we demonstrate that these effects can be explained by PV and SST interneurons having preferential contributions to feedforward and feedback inhibition, respectively. Our findings demonstrate that distinct subtypes of inhibitory interneurons have frequency-selective roles in the spatiotemporal synchronization of precise spike times.

Dissociation of somatostatin and parvalbumin interneurons circuit dysfunctions underlying hippocampal theta and gamma oscillations impaired by amyloid ß oligomers in vivo.

Accumulation of amyloid ß oligomers (AßO) in Alzheimer's disease (AD) impairs hippocampal theta and gamma oscillations. These oscillations are important in memory functions and depend on distinct subtypes of hippocampal interneurons such as somatostatin-positive (SST) and parvalbumin-positive (PV) interneurons. Here, we investigated whether AßO causes dysfunctions in SST and PV interneurons by optogenetically manipulating them during theta and gamma oscillations in vivo in AßO-injected SST-Cre or PV-Cre mice. Hippocampal in vivo multi-electrode recordings revealed that optogenetic activation of channelrhodopsin-2 (ChR2)-expressing SST and PV interneurons in AßO-injected mice selectively restored AßO-induced reduction of the peak power of theta and gamma oscillations, respectively, and resynchronized CA1 pyramidal cell (PC) spikes. Moreover, SST and PV interneuron spike phases were resynchronized relative to theta and gamma oscillations, respectively. Whole-cell voltage-clamp recordings in CA1 PC in ex vivo hippocampal slices from AßO-injected mice revealed that optogenetic activation of SST and PV interneurons enhanced spontaneous inhibitory postsynaptic currents (IPSCs) selectively at theta and gamma frequencies, respectively. Furthermore, analyses of the stimulus-response curve, paired-pulse ratio, and short-term plasticity of SST and PV interneuron-evoked IPSCs ex vivo showed that AßO increased the initial GABA release probability to depress SST/PV interneuron's inhibitory input to CA1 PC selectively at theta and gamma frequencies, respectively. Our results reveal frequency-specific and interneuron subtype-specific presynaptic dysfunctions of SST and PV interneurons' input to CA1 PC as the synaptic mechanisms underlying AßO-induced impairments of hippocampal network oscillations and identify them as potential therapeutic targets for restoring hippocampal network oscillations in early AD.

CalibraCurve: A Tool for Calibration of Targeted MS-Based Measurements.

Targeted proteomics techniques allow accurate quantitative measurements of analytes in complex matrices with dynamic linear ranges that span up to 4-5 orders of magnitude. Hence, targeted methods are promising for the development of robust protein assays in several sensitive areas, for example, in health care. However, exploiting the full method potential requires reliable determination of the dynamic range along with related quantification limits for each analyte. Here, a software named CalibraCurve that enables an automated batch-mode determination of dynamic linear ranges and quantification limits for both targeted proteomics and similar assays is presented. The software uses a variety of measures to assess the accuracy of the calibration, namely precision and trueness. Two different kinds of customizable graphs are created (calibration curves and response factor plots). The accuracy measures and the graphs offer an intuitive, detailed, and reliable opportunity to assess the quality of the model fit. Thus, CalibraCurve is deemed a highly useful and flexible tool to facilitate the development and control of reliable SRM/MRM-MS-based proteomics assays.

Optogenetic activation of parvalbumin and somatostatin interneurons selectively restores theta-nested gamma oscillations and oscillation-induced spike timing-dependent long-term potentiation impaired by amyloid ß oligomers.

BACKGROUND: Abnormal accumulation of amyloid ß1-42 oligomers (AßO1-42), a hallmark of Alzheimer's disease, impairs hippocampal theta-nested gamma oscillations and long-term potentiation (LTP) that are believed to underlie learning and memory. Parvalbumin-positive (PV) and somatostatin-positive (SST) interneurons are critically involved in theta-nested gamma oscillogenesis and LTP induction. However, how AßO1-42 affects PV and SST interneuron circuits is unclear. Through optogenetic manipulation of PV and SST interneurons and computational modeling of the hippocampal neural circuits, we dissected the contributions of PV and SST interneuron circuit dysfunctions on AßO1-42-induced impairments of hippocampal theta-nested gamma oscillations and oscillation-induced LTP. RESULTS: Targeted whole-cell patch-clamp recordings and optogenetic manipulations of PV and SST interneurons during in vivo-like, optogenetically induced theta-nested gamma oscillations in vitro revealed that AßO1-42 causes synapse-specific dysfunction in PV and SST interneurons. AßO1-42 selectively disrupted CA1 pyramidal cells (PC)-to-PV interneuron and PV-to-PC synapses to impair theta-nested gamma oscillogenesis. In contrast, while having no effect on PC-to-SST or SST-to-PC synapses, AßO1-42 selectively disrupted SST interneuron-mediated disinhibition to CA1 PC to impair theta-nested gamma oscillation-induced spike timing-dependent LTP (tLTP). Such AßO1-42-induced impairments of gamma oscillogenesis and oscillation-induced tLTP were fully restored by optogenetic activation of PV and SST interneurons, respectively, further supporting synapse-specific dysfunctions in PV and SST interneurons. Finally, computational modeling of hippocampal neural circuits including CA1 PC, PV, and SST interneurons confirmed the experimental observations and further revealed distinct functional roles of PV and SST interneurons in theta-nested gamma oscillations and tLTP induction. CONCLUSIONS: Our results reveal that AßO1-42 causes synapse-specific dysfunctions in PV and SST interneurons and that optogenetic modulations of these interneurons present potential therapeutic targets for restoring hippocampal network oscillations and synaptic plasticity impairments in Alzheimer's disease.

Ultraviolet Fabry-Perot cavity with stable finesse under ultrahigh vacuum conditions.

We have constructed an apparatus containing a linear ion trap and a high-finesse optical cavity in the ultraviolet spectral range. In our construction, we have avoided all organic materials inside the ultrahigh vacuum chamber. We show that, unlike previously reported, the optical cavity does not degrade in performance over a time scale of 9 months.

Quasi-simultaneous multiplane calcium imaging of neuronal circuits.

Two-photon excitation fluorescence microscopy is widely used to study the activity of neuronal circuits. However, the fast imaging is typically constrained to a single lateral plane for a standard microscope design. Given that cortical neuronal networks in a mouse brain are complex three-dimensional structures organised in six histologically defined layers which extend over many hundreds of micrometres, there is a strong demand for microscope systems that can record neuronal signalling in volumes. Henceforth, we developed a quasi-simultaneous multiplane imaging technique combining an acousto-optic deflector and static remote focusing to provide fast imaging of neurons from different axial positions inside the cortical layers without the need for mechanical disturbance of either the objective lens or the specimen. The hardware and the software are easily adaptable to existing two-photon microscopes. Here, we demonstrated that our imaging method can record, at high speed and high image contrast, the calcium dynamics of neurons in two different imaging planes separated axially with the in-focus and the refocused planes 120 µm and 250 µm below the brain surface respectively.