Diving into the brain: deep-brain imaging techniques in conscious animals

Abstract

<jats:p>In most species, survival relies on the hypothalamic control of endocrine axes that regulate critical functions such as reproduction, growth, and metabolism. For decades, the complexity and inaccessibility of the hypothalamic–pituitary axis has prevented researchers from elucidating the relationship between the activity of endocrine hypothalamic neurons and pituitary hormone secretion. Indeed, the study of central control of endocrine function has been largely dominated by ‘traditional’ techniques that consist of studying <jats:italic>in vitro</jats:italic> or <jats:italic>ex vivo</jats:italic> isolated cell types without taking into account the complexity of regulatory mechanisms at the level of the brain, pituitary and periphery. Nowadays, by exploiting modern neuronal transfection and imaging techniques, it is possible to study hypothalamic neuron activity <jats:italic>in situ</jats:italic>, in real time, and in conscious animals. Deep-brain imaging of calcium activity can be performed through gradient-index lenses that are chronically implanted and offer a ‘window into the brain’ to image multiple neurons at single-cell resolution. With this review, we aim to highlight deep-brain imaging techniques that enable the study of neuroendocrine neurons in awake animals whilst maintaining the integrity of regulatory loops between the brain, pituitary and peripheral glands. Furthermore, to assist researchers in setting up these techniques, we discuss the equipment required and include a practical step-by-step guide to performing these deep-brain imaging studies.</jats:p>

A luciferase prosubstrate and a red bioluminescent calcium indicator for imaging neuronal activity in mice

“…Fluorescence imaging is the standard method for following brain activity in small behaving animals. Genetically encoded fluorescent indicators, including GCaMPs and other Ca 2+ , voltage, and neurotransmitter indicators, allowed the tracking of neuronal activities in specific brain regions and cell types in mammals with the high spatiotemporal resolution for extended periods 1 – 3 . Despite the progress, fluorescence neuronal imaging is practically invasive and only reaches a shallow depth.…”

A luciferase prosubstrate and a red bioluminescent calcium indicator to image neuronal activity

“…Genetically encoded fluorescent indicators, including GCaMPs and other Ca 2+ , voltage, neurotransmitter indicators, allowed the tracking of neuronal activities of specific brain regions and cell types in mammals with a high spatiotemporal resolution for extended periods. [1][2][3] Despite the progress, fluorescence neuronal imaging is practically invasive and only reaches a shallow depth. Cranial windows or thinned skulls are often used to access the cortex.…”

“…Due to tissue absorption and scattering, the imaging depth is ~ 200 μm for widefield one-photon and a couple of millimeters with multiphoton excitation. [3][4][5] To reach deeper brain regions, more invasive procedures, such as implanting optical fibers or gradient-index (GRIN) lenses, are needed. 3 Bioluminescence, which refers to photon emission from luciferase-catalyzed exothermic oxidation of the corresponding luciferin, is a promising imaging modality for noninvasive in vivo recording.…”

“…[3][4][5] To reach deeper brain regions, more invasive procedures, such as implanting optical fibers or gradient-index (GRIN) lenses, are needed. 3 Bioluminescence, which refers to photon emission from luciferase-catalyzed exothermic oxidation of the corresponding luciferin, is a promising imaging modality for noninvasive in vivo recording. 6,7 Because bioluminescence needs no excitation, photons emitting from the embedded light sources can travel through several centimeters of mammalian tissue.…”

“…Ca 2+ is a ubiquitous second messenger and intracellular Ca 2+ has been used as a proxy for neuronal activity. [1][2][3] Previous studies have used aequorin, a Ca 2+ -sensitive photoprotein, and its mutants for Ca 2+ detection, but they all emit photons very slowly. 16,17 Other studies introduced Ca 2+ -sensory elements into luciferases, including NanoLuc and NanoLuc-FP hybrid reporters, resulting in Ca 2+ indicators with much-improved light production rates.…”