Neurons communicate through neurotransmission at highly specialized junctions called synapses. Each neuron forms numerous synaptic connections, consisting of presynaptic and postsynaptic terminals. Upon the arrival of an action potential, neurotransmitters are released from the presynaptic site and diffuse across the synaptic cleft to bind specialized receptors at the postsynaptic terminal. This process is tightly regulated by several proteins at both presynaptic and postsynaptic sites. The localization, abundance, and function of these proteins are essential for productive neurotransmission and are often affected in neurological and neurodegenerative disorders. Here, we outline a method for purifying mouse synaptosomes and using limited tryptic digestion to assess the subcellular localization of synaptic proteins. During synaptosomes purification, presynaptic terminals reseal and are protected from proteolysis, while postsynaptic proteins remain susceptible to tryptic cleavage. These changes can easily be evaluated by western blot analysis. This approach offers a straightforward and reliable method to evaluate the subcellular localization of synaptic proteins based on their proteolytic sensitivity, providing valuable insights into synaptic physiology and pathology.

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).

Intracellular signaling pathways directly and indirectly regulate neuronal activity. In cellular electrophysiological measurements with sharp electrodes or whole-cell patch clamp recordings, there is a great risk that these signaling pathways are disturbed, significantly altering the electrophysiological properties of the measured neurons. Perforated-patch clamp recordings circumvent this issue, allowing long-term electrophysiological recordings with minimized impairment of the intracellular milieu. Based on previous studies, we describe a superstition-free protocol that can be used to routinely perform perforated patch clamp recordings for current and voltage measurements.

The Substantia Nigra pars compacta (SNc) is a midbrain dopaminergic nucleus that plays a key role in modulating motor and cognitive functions. It is crucially involved in several disorders, particularly Parkinson’s disease, which is characterized by a progressive loss of SNc dopaminergic cells. Electrophysiological studies on SNc neurons are of paramount importance to understand the role of dopaminergic transmission in health and disease. Here, we provide an extensive protocol to prepare SNc-containing mouse brain slices and record the electrical activity of dopaminergic cells. We describe all the necessary steps, including mouse transcardiac perfusion, brain extraction, slice cutting, and patch-clamp recordings.

The Drosophila retina contains light-sensitive photoreceptors (R cells) with distinct spectral sensitivities that allow them to distinguish light by its spectral composition. R7 and R8 photoreceptors are important for color vision, and can be further classified into pale (p) or yellow (y) subtypes depending on the rhodopsin expressed. While both R7y and R7p are sensitive to UV light, R8y and R8p detect light in the green and blue spectrum, respectively. The ability of R7 and R8 photoreceptors to distinguish different spectral sensitivities and the natural preference for Drosophila towards light sources (phototaxis), allow for the development of a phototactic T-maze assay that compares the functionality of different R7 and R8 subtypes. A “UV vs. blue” choice can compare the functionalities of R7p and R8p photoreceptors, while a “UV vs. green” choice can compare the functionalities of R7y and R8y photoreceptors. Additionally, a “blue vs. green” choice could be used to compare R8p and R8y photoreceptors, while a “dark vs. light" choice could be used to determine overall vision functionality. Although electrophysiological recordings and calcium imaging have been used to examine functionality of R7 and R8 photoreceptors, these approaches require expensive equipment and are technically challenging. The phototactic T-maze assay we present here is a robust, straight-forward and an inexpensive method to study genetic and developmental factors that contribute to the individual functionality of R7 and R8 photoreceptors, and is especially useful when performing large-scale genetic screens.

Sleep is a conserved neurobehavioral state observed in animals with sufficiently complex nervous systems and is critical for survival. While the exact function of sleep remains unknown, the lack of sleep can have a range of physiological and behavioral effects. Studies in invertebrates and vertebrates have identified conserved neural mechanisms and cellular pathways in control of sleep, wakefulness and arousal. Methodologies to measure sleep have ranged from EEG recordings in humans and rodents to in-depth analysis of locomotor patterns in flies, fish and worms. Here we focus on sleep measurements using activity monitoring in the highly versatile experimental model system, Drosophila melanogaster, which is amenable to a number of genetic, physiological and behavioral manipulations. Further, we also describe methods used to manipulate sleep and wakefulness to understand the neural regulation of sleep and how organisms balance sleep, wakefulness and behavioral arousal. Sleep as a behavioral state is regulated by a number of factors including food, environmental conditions, and genetic background. The methodologies described here provide, a high-throughput approach to study neural regulation of sleep and factors that affect this complex behavior.

Microglia are the resident immune cells of the central nervous system (CNS). In the last year, the improvements in the transgenic mouse technologies and imaging techniques have shed light on microglia functions under physiological conditions. Microglia continuously scan the brain parenchyma with their highly motile processes, maintaining tissue homeostasis and participating in neuronal circuits refinement. Here, we describe a protocol that enables us to perform time-lapse imaging of microglial cells in acute hippocampal slices, making image acquisition possible on an electrophysiology rig equipped with a standard imaging system. Using this ex vivo approach, we investigated microglial processes scanning abilities under physiological condition in hippocampus.

Presynaptic boutons at nerve terminals are densely packed with synaptic vesicles, specialized organelles for rapid and regulated neurotransmitter secretion. Upon depolarization of the nerve terminal, synaptic vesicles fuse at specializations called active zones that are localized at discrete compartments in the plasma membrane to initiate synaptic transmission. A small proportion of synaptic vesicles are docked and primed for immediate fusion upon synaptic stimulation, which together comprise the readily releasable pool. The size of the readily releasable pool is an important property of synapses, which influences release probability and can dynamically change during various forms of plasticity. Here we describe a detailed protocol for estimating the readily releasable pool at a model glutamatergic synapse, the Drosophila neuromuscular junction. This synapse is experimentally robust and amenable to sophisticated genetic, imaging, electrophysiological, and pharmacological approaches. We detail the experimental design, electrophysiological recording procedure, and quantitative analysis necessary to determine the readily releasable pool size. This technique requires the use of a two-electrode voltage-clamp recording configuration in elevated external Ca²⁺ with high frequency stimulation. We have used this assay to measure the readily releasable pool size and reveal that a form of homeostatic plasticity modulates this pool with synapse-specific and compartmentalized precision. This powerful approach can be utilized to illuminate the dynamics of synaptic vesicle trafficking and plasticity and determine how synaptic function adapts and deteriorates during states of altered development, stress and neuromuscular disease.

In songbirds and higher mammals, early auditory experience during childhood is critical to detect and discriminate sound patterns in adulthood. However, the neural and molecular nature of this acquired ability remains elusive. Here, we describe a new behavioral paradigm with Drosophila melanogaster to investigate how the auditory experience shapes sound perception. This behavioral paradigm consists of two parts: training session and test session. In the training session, we keep the flies singly in a training capsule and expose them to training sound for 6 days after eclosion. After the training session, flies are subjected to the test session, in which the mating behaviors of flies are monitored upon sound playback. As the training and test sounds, we use two types of artificial sound, which correspond to the pattern of conspecific and heterospecific courtship songs of fruit flies. By applying this method, we can measure how the acoustic experience with the conspecific song as a young adult sharpens the song preference and mate selection as a breeding adult in the fruit fly.