Protocols in Current Issue
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0 Q&A 749 Views May 5, 2023

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.

0 Q&A 6914 Views Mar 5, 2018
In this protocol, we introduce an effective method for voltage-sensitive dye (VSD) loading and imaging of leech ganglia as used in Tomina and Wagenaar (2017). Dissection and dye loading procedures are the most critical steps toward successful whole-ganglion VSD imaging. The former entails the removal of the sheath that covers neurons in the segmental ganglion of the leech, which is required for successful dye loading. The latter entails gently flowing a new generation VSD, VF2.1(OMe).H, onto both sides of the ganglion simultaneously using a pair of peristaltic pumps. We expect the described techniques to translate broadly to wide-field VSD imaging in other thin and relatively transparent nervous systems.
1 Q&A 9078 Views Sep 20, 2017
The proof of concept for bioluminescence monitoring of neural activity in zebrafish with the genetically encoded calcium indicator GFP-aequorin has been previously described (Naumann et al., 2010) but challenges remain. First, bioluminescence signals originating from a single muscle fiber can constitute a major pitfall. Second, bioluminescence signals emanating from neurons only are very small. To improve signals while verifying specificity, we provide an optimized 4 steps protocol achieving: 1) selective expression of a zebrafish codon-optimized GFP-aequorin, 2) efficient soaking of larvae in GFP-aequorin substrate coelenterazine, 3) bioluminescence monitoring of neural activity from motor neurons in free-tailed moving animals performing acoustic escapes and 4) verification of the absence of muscle expression using immunohistochemistry.
1 Q&A 13924 Views Feb 5, 2016
Cortico-cortical interactions play crucial roles in various brain functions. Here, we present a detailed surgical procedure for cortical voltage-sensitive dye (VSD) imaging that allows monitoring of spatiotemporal dynamics in cortical activity in living mice. Cortical neurons in the upper layers (layer 1-3) are stained with a VSD, and an image sensor with a fast sampling rate (500 Hz) detects fluorescent changes in corrective activity. The procedure includes fixing a mouse brain to a stereotaxic apparatus, craniotomy on a large cortical area, VSD staining, and wide-field imaging of cortical activity. The entire procedure can be completed in 5 h (from the administration of anesthesia to the start of cortical VSD imaging).
0 Q&A 11079 Views Dec 5, 2014
This protocol comprises the entire process of fluorescent measurement of vesicle recycling using the probe SynaptopHluorin, a pH-dependent GFP variant whose fluorescence increases at the synapse upon vesicle release due to fluorescence quenching in acidic vesicles. This technique provides a genetic tool to monitor synaptic vesicle recycling in real time in cultured hippocampal neurons.
0 Q&A 11665 Views Aug 5, 2013
This protocol describes how to visualize neuronal morphology and how to determine neuronal complexity of immature and mature hippocampal neurons in the mouse in vivo including tissue preparation, staining of brain sections and confocal cell analysis.
0 Q&A 16069 Views Jul 20, 2012
Microfluidics chamber is an ideal tool to study local events that occurring in neuronal projections by perfectly compartmentalizing the cell soma from certain branches. It is very well suited for live cell imaging or immunohistochemistry staining. This protocol has been carefully modified in detail to fit the requirement of primary rat hippocampal neuronal cultures. It can also be applied to a more general neuronal culture purpose in microfluidics.

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