神经科学

Stimulus-induced narrow-band gamma oscillations (20–70 Hz) are induced in the visual areas of the brain when particular visual stimuli, such as bars, gratings, or full-screen hue, are shown to the subject. Such oscillations are modulated by higher cognitive functions, like attention, and working memory, and have been shown to be abnormal in certain neuropsychiatric disorders, such as schizophrenia, autism, and Alzheimer’s disease. However, although electroencephalogram (EEG) remains one of the most non-invasive, inexpensive, and accessible methods to record brain signals, some studies have failed to observe discernable gamma oscillations in human EEG. In this manuscript, we have described in detail a protocol to elicit robust gamma oscillations in human EEG. We believe that our protocol could help in developing non-invasive gamma-based biomarkers in human EEG, for the early detection of neuropsychiatric disorders.

Determining the neuronal circuitry responsible for specific behaviors is a major focus in the field of neurobiology. Activity-dependent immediate early genes (IEGs), transcribed and translated shortly after neurons discharge action potentials, have been used extensively to either identify or gain genetic access to neurons and brain regions involved in such behaviors. By using immunohistochemistry for the protein product of the IEG c-Fos combined with retrograde labeling of specific neuronal populations, precise experimental timing, and identical data acquisition and processing, we present a method to quantitatively identify specific neuronal subpopulations that were active during social encounters. We have previously used this method to show a stronger recruitment of ventral hippocampal neurons that project to the medial prefrontal cortex, compared to those that project to the lateral hypothalamus, following social interactions. After optimization of surgeries for the injection of retrograde tracers, this method will be useful for the identification and mapping of neuronal populations engaged in many different behaviors.

Activation of type 1 cannabinoid (CB₁) receptors by endogenous, exogenous (cannabis derivatives) or synthetic cannabinoids (i.e., CP 55.940, Win-2) has a wide variety of behavioral effects due to the presence of CB₁ receptors in the brain. In situ hybridization and immunohistochemical techniques have been crucial for defining the CB₁ receptor expression and localization at the cellular level. Nevertheless, more advanced methods are needed to reveal the precise topography of CB₁ receptors in the brain, especially in unsuspected sites such as other cell types and organelles with low receptor expression (e.g., glutamatergic neurons, astrocytes, mitochondria). High-resolution immunoelectron microscopy provides a more precise detection method for the subcellular localization of CB₁ receptors in the brain. Herein, we describe a single pre-embedding immunogold method for electron microscopy based on the use of specific CB₁ receptor antibodies and silver-intensified 1.4 nm gold-labeled Fab' fragments, and a combined pre-embedding immunogold and immunoperoxidase method that employs biotinylated secondary antibodies and avidin-biotin-peroxidase complex for the simultaneous localization of CB₁ receptors and protein markers of specific brain cells or synapses (e.g., GFAP, GLAST, IBA-1, PSD-95, gephyrin). In addition, a post-embedding immunogold method is also described and compared to the pre-embedding labeling procedure. These methods provide a relatively easy and useful approach for revealing the subcellular localization of low amounts of CB₁ receptors in glutamatergic synapses, astrocytes, neuronal and astrocytic mitochondria in the brain.

The hypothalamus is a primary brain area which, in mammals, regulates several physiological functions that are all related to maintaining general homeostasis, by linking the central nervous system (CNS) and the periphery. The hypothalamus itself can be considered an endocrine brain region of some sort as it hosts in its different nuclei several kinds of neuropeptide-producing and -secreting neurons. These neuropeptides have specific roles and participate in the regulation of homeostasis in general, which includes the regulation of energy metabolism, feeding behavior, water intake and body core temperature for example.

As previously mentioned, in order to exert their effects, these peptides have to be produced but also, and mostly, to be secreted. In this context, it is of great importance to be able to assess how certain conditions, diseases, or treatments can actually influence the secretion of neuropeptides, thus the function of the different neuropeptidergic circuits.

One method to assess this is the perifusion of hypothalamic explants followed by quantification of peptides within the collected fractions.

Here, we explain step-by-step how to collect fractions during ex vivo perifusion of hypothalamic explants in which one can determine quantitatively neuropeptide/neurohormone release from these viable isolated tissues. Hypothalami perifusion has two great advantages over other existing assays: (1) it allows pharmacological manipulation to dissect out signaling mechanisms underlying release of different neuropeptides/neurohormones in the hypothalamic explants and, (2) it allows simultaneous experiments with different conditions on multiple hypothalami preparations, (3) it is, to our knowledge, the only method that permits the study of neuropeptide secretion in basal conditions and under repeated stimulations with the same hypothalami explants.

Action potential conduction velocity is the speed at which an action potential (AP) propagates along an axon. Measuring AP conduction velocity is instrumental in determining neuron health, function, and computational capability, as well as in determining short-term dynamics of neuronal communication and AP initiation (Ballo and Bucher, 2009; Bullock, 1951; Meeks and Mennerick, 2007; Rosenthal and Bezanilla, 2000; Städele and Stein, 2016; Swadlow and Waxman, 1976). Conduction velocity can be measured using extracellular recordings along the nerve through which the axon projects. Depending on the number of axons in the nerve, AP velocities of individual or many axons can be detected.

This protocol outlines how to measure AP conduction velocity of (A) stimulated APs and (B) spontaneously generated APs by using two spatially distant extracellular electrodes. Although an invertebrate nervous system is used here, the principles of this technique are universal and can be easily adjusted to other nervous system preparations (including vertebrates).