Editor
Geoffrey C. Y. Lau
  • Faculty, Department of Neuroscience, City University of Hong Kong
Research fields
  • Neuroscience
Semi-Automated Assessment of Long-Term Olfactory Habituation in Drosophila melanogaster Using the Olfactory Arena
Authors:  Camilla Roselli, Mani Ramaswami and Marcia M. Aranha, date: 11/05/2024, view: 363, Q&A: 0

Long-lasting memories are a core aspect of an animal’s life. Such memories are characterized by unique molecular mechanisms and often unique circuitry, neither of which are completely understood in vivo. The deep knowledge of the identity and connectivity of neurons of the fruit fly Drosophila melanogaster, as well as the sophisticated genetic tools that allow in vivo perturbations and physiology monitoring, make it a remarkably useful organism in which to investigate the molecular mechanisms of long-term memories. In this protocol, we focus on habituation, a non-associative form of learning, and describe a reliable, semi-automated technique to induce and assess long-term olfactory habituation (LTH) in Drosophila using the olfactory arena, thus providing a method aligned with recent technological progress in behavioral measurement. Prior work has shown that LTH is induced by a 4-day exposure to an odorant and is characterized by a long-lasting (> 24 h) reduction in behavioral response to the exposed odorant, measured using a manual and skill-intensive Y-maze assay. Here, we present a semi-automated protocol for obtaining quantifiable measures of LTH, at the level of detail required for other investigators in the field. Unlike previously described methods, the protocol presented here provides quantitative and detailed behavioral measurements obtained by video recording that can be shared with the scientific community and allows sophisticated forms of offline analysis. We suggest that this procedure has the potential to advance our understanding of molecular and circuit mechanisms of olfactory habituation, its control via neuromodulation, and its interactions with other forms of memory.

Flow Cytometry Analysis of Microglial Phenotypes in the Murine Brain During Aging and Disease

Microglia, the brain's primary resident immune cell, exists in various phenotypic states depending on intrinsic and extrinsic signaling. Distinguishing between these phenotypes can offer valuable biological insights into neurodevelopmental and neurodegenerative processes. Recent advances in single-cell transcriptomic profiling have allowed for increased granularity and better separation of distinct microglial states. While techniques such as immunofluorescence and single-cell RNA sequencing (scRNA-seq) are available to differentiate microglial phenotypes and functions, these methods present notable limitations, including challenging quantification methods, high cost, and advanced analytical techniques. This protocol addresses these limitations by presenting an optimized cell preparation procedure that prevents ex vivo activation and a flow cytometry panel to distinguish four distinct microglial states from murine brain tissue. Following cell preparation, fluorescent antibodies were applied to label 1) homeostatic, 2) disease-associated (DAM), 3) interferon response (IRM), and 4) lipid-droplet accumulating (LDAM) microglia, based on gene markers identified in previous scRNA-seq studies. Stained cells were analyzed by flow cytometry to assess phenotypic distribution as a function of age and sex. A key advantage of this procedure is its adaptability, allowing the panel provided to be enhanced using additional markers with an appropriate cell analyzer (i.e., Cytek Aurora 5 laser spectral flow cytometer) and interrogating different brain regions or disease models. Additionally, this protocol does not require microglial cell sorting, resulting in a relatively quick and straightforward experiment. Ultimately, this protocol can compare the distribution of microglial phenotypic states between various experimental groups, such as disease state or age, with a lower cost and higher throughput than scRNA-seq.

Unlocking Bio-Instructive Polymers: A Novel Multi-Well Screening Platform Based on Secretome Sampling
Authors:  Shirin Fateh, Reem A. Alromaihi, Amir M. Ghaemmaghami and Morgan R. Alexander, date: 02/20/2024, view: 1298, Q&A: 0

Biomaterials are designed to interact with biological systems to replace, support, enhance, or monitor their function. However, there are challenges associated with traditional biomaterials’ development due to the lack of underlying theory governing cell response to materials’ chemistry. This leads to the time-consuming process of testing different materials plus the adverse reactions in the body such as cytotoxicity and foreign body response. High-throughput screening (HTS) offers a solution to these challenges by enabling rapid and simultaneous testing of a large number of materials to determine their bio-interactions and biocompatibility. Secreted proteins regulate many physiological functions and determine the success of implanted biomaterials through directing cell behaviour. However, the majority of biomaterials’ HTS platforms are suitable for microscopic analyses of cell behaviour and not for investigating non-adherent cells or measuring cell secretions. Here, we describe a multi-well platform adaptable to robotic printing of polymers and suitable for secretome profiling of both adherent and non-adherent cells. We detail the platform's development steps, encompassing the preparation of individual cell culture chambers, polymer printing, and the culture environment, as well as examples to demonstrate surface chemical characterisation and biological assessments of secreted mediators. Such platforms will no doubt facilitate the discovery of novel biomaterials and broaden their scope by adapting wider arrays of cell types and incorporating assessments of both secretome and cell-bound interactions.


Key features

• Detailed protocols for preparation of substrate for contact printing of acrylate-based polymers including O2 plasma etching, functionalisation process, and Poly(2-hydroxyethyl methacrylate) (pHEMA) dip coating.

• Preparations of 7 mm × 7 mm polymers employing pin printing system.

• Provision of confined area for each polymer using ProPlate® multi-well chambers.

• Compatibility of this platform was validated using adherent cells [primary human monocyte–derived macrophages (MDMs)) and non-adherent cells (primary human monocyte–derived dendritic cells (moDCs)].

• Examples of the adaptability of the platform for secretome analysis including five different cytokines using enzyme-linked immunosorbent assay (ELISA, DuoSet®).


Graphical overview


Headplate Installation and Craniotomy for Awake In Vivo Electrophysiological Recordings or Two-Photon Imaging of the Mouse Inferior Colliculus
Authors:  Blom Kraakman, Sofja Solovjova, J. Gerard G. Borst and Aaron Benson Wong, date: 12/20/2023, view: 600, Q&A: 0

The inferior colliculus (IC) is an important processing center in the auditory system, which also receives non-auditory sensory input. The IC consists of several subnuclei whose functional role in (non-) auditory processing and plastic response properties are best approached by studying awake animals, preferably in a longitudinal fashion. The increasing use of mice in auditory research, the availability of genetic models, and the superficial location of the IC in the mouse have made it an attractive species for studying IC function. Here, we describe a protocol for exposing the mouse IC for up to a few weeks for in vivo imaging or electrophysiology in a stable manner. This method allows for a broader sampling of the IC while maintaining the brain surface in good quality and without reopening the craniotomy. Moreover, as it is adaptable for both electrophysiological recordings of the entire IC and imaging of the dorsal IC surface, it can be applied to answer a multitude of questions.


Key features

• A surgical protocol for long-term physiological recordings from the same or separate neuronal populations in the inferior colliculus.

• Optimized for awake in vivo experiments in the house mouse (Mus musculus).

Testing for Allele-specific Expression from Human Brain Samples

Many single nucleotide polymorphisms (SNPs) identified by genome-wide association studies exert their effects on disease risk as expression quantitative trait loci (eQTL) via allele-specific expression (ASE). While databases for probing eQTLs in tissues from normal individuals exist, one may wish to ascertain eQTLs or ASE in specific tissues or disease-states not characterized in these databases. Here, we present a protocol to assess ASE of two possible target genes (GPNMB and KLHL7) of a known genome-wide association study (GWAS) Parkinson’s disease (PD) risk locus in postmortem human brain tissue from PD and neurologically normal individuals. This was done using a sequence of RNA isolation, cDNA library generation, enrichment for transcripts of interest using customizable cDNA capture probes, paired-end RNA sequencing, and subsequent analysis. This method provides increased sensitivity relative to traditional bulk RNAseq-based and a blueprint that can be extended to the study of other genes, tissues, and disease states.


Key features

• Analysis of GPNMB allele-specific expression (ASE) in brain lysates from cognitively normal controls (NC) and Parkinson’s disease (PD) individuals.

• Builds on the ASE protocol of Mayba et al. (2014) and extends application from cells to human tissue.

• Increased sensitivity by enrichment for desired transcript via RNA CaptureSeq (Mercer et al., 2014).

• Optimized for human brain lysates from cingulate gyrus, caudate nucleus, and cerebellum.


Graphical overview


Simultaneous Microendoscopic Calcium Imaging and EEG Recording of Mouse Brain during Sleep
Authors:  Sasa Teng and Yueqing Peng, date: 05/05/2023, view: 906, Q&A: 0

Sleep is a conserved biological process in the animal kingdom. Understanding the neural mechanisms underlying sleep state transitions is a fundamental goal of neurobiology, important for the development of new treatments for insomnia and other sleep-related disorders. Yet, brain circuits controlling this process remain poorly understood. A key technique in sleep research is to monitor in vivo neuronal activity in sleep-related brain regions across different sleep states. These sleep-related regions are usually located deeply in the brain. Here, we describe technical details and protocols for in vivo calcium imaging in the brainstem of sleeping mice. In this system, sleep-related neuronal activity in the ventrolateral medulla (VLM) is measured using simultaneous microendoscopic calcium imaging and electroencephalogram (EEG) recording. By aligning calcium and EEG signals, we demonstrate that VLM glutamatergic neurons display increased activity during the transition from wakefulness to non-rapid eye movement (NREM) sleep. The protocol described here can be applied to study neuronal activity in other deep brain regions involved in REM or NREM sleep.

Parasitoid Wasp Culturing and Assay to Study Parasitoid-induced Reproductive Modifications in Drosophila
Authors:  Madhumala K. Sadanandappa, Shivaprasad H. Sathyanarayana and Giovanni Bosco, date: 01/05/2023, view: 610, Q&A: 0

In nature, parasitoid wasp infections are a major cause of insect mortality. Parasitoid wasps attack a vast range of insect species to lay their eggs. As a defense, insects evolved survival strategies to protect themselves from parasitoid infection. While a growing number of studies reported both host defensive tactics and parasitoid counter-offensives, we emphasize that this parasite–host relationship presents a unique ecological and evolutionary relevant model that is often challenging to replicate in a laboratory. Although maintaining parasitoid wasp cultures in the laboratory requires meticulous planning and can be labor intensive, a diverse set of wasp species that target many different insect types can be maintained in similar culture conditions. Here, we describe the protocol for culturing parasitoid wasp species on Drosophila larvae and pupae in laboratory conditions. We also detail an egg-laying assay to assess the reproductive modification of Drosophila females in response to parasitoid wasps. This behavioral study is relatively simple and easily adaptable to study environmental or genetic influences on egg-laying, a readout for female germline development. Neither the parasitoid culture conditions or the behavioral assay require special supplies or equipment, making them a powerful and versatile approach in research or teaching laboratory settings.


Graphical abstract


Fluorescence Screens for Identifying Central Nervous System–Acting Drug–Biosensor Pairs for Subcellular and Supracellular Pharmacokinetics

Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)–acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit pairs comprising a drug and biosensor, with each screen including 13–18 candidate biosensors and 44–84 candidate drugs. After a favorable hit pair is identified and validated via these protocols, the biosensor is then optimized, as described in other papers, for sensitivity and selectivity to the drug. We also show sample hit pair data that may lead to future intensity-based drug-sensing fluorescent reporters (iDrugSnFRs). These protocols will assist scientists to use fluorescence responses as criteria in identifying favorable fluorescent biosensor variants for CNS-acting drugs that presently have no corresponding biosensor partner.


Graphical abstract:




Trichloroacetic Acid Fixation and Antibody Staining of Zebrafish Larvae
Authors:  E. Anne Martin, Sundas Ijaz, Alberto E. Pereda and Adam C. Miller, date: 01/20/2022, view: 2521, Q&A: 0

Larval zebrafish have been established as an excellent model for examining vertebrate biology, with many researchers using the system for neuroscience. Controlling a fast escape response of the fish, the Mauthner cells and their associated network are an attractive model, given their experimental accessibility and fast development, driving ethologically relevant behavior in the first five days of development. Here, we describe methods for immunostaining electrical and chemical synapse proteins at 3-7 days post fertilization (dpf) in zebrafish using tricholoracetic acid fixation. The methods presented are ideally suited to easily visualize neural circuits and synapses within the fish.


Subcellular RNA-seq for the Analysis of the Dendritic and Somatic Transcriptomes of Single Neurons
Authors:  Julio D. Perez and Erin M. Schuman, date: 01/05/2022, view: 3219, Q&A: 0

In neurons, local translation in dendritic and axonal compartments allows for the fast and on-demand modification of the local proteome. As the last few years have witnessed dramatic advancements in our appreciation of the brain’s neuronal diversity, it is increasingly relevant to understand how local translation is regulated according to cell type. To this end, both sequencing-based and imaging-based techniques have recently been reported. Here, we present a subcellular single cell RNA sequencing protocol that allows molecular quantification from the soma and dendrites of single neurons, and which can be scaled up for the characterization of several hundreds to thousands of neurons. Somata and dendrites of cultured neurons are dissected using laser capture microdissection, followed by cell lysis to release mRNA content. Reverse transcription is then conducted using an indexed primer that allows the downstream pooling of samples. The pooled cDNA library is prepared for and sequenced in an Illumina platform. Finally, the data generated are processed and converted into a gene vs. cells digital expression table. This protocol provides detailed instructions for both wet lab and bioinformatic steps, as well as insights into controls, data analysis, interpretations, and ways to achieve robust and reproducible results.


Graphic abstract:



Subcellular Single Cell RNA-seq in Neurons.

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