Technological developments in genetics and systems neuroscience have enabled specific neurons to be manipulated in vivo. The tools of chemical genetics and optogenetics make it possible to study the hypothalamus. Deep-brain imaging techniques enable the study of endogenous brain activity in neuroendocrine populations in awake animals whilst maintaining the integrity of regulatory loops between the brain, pituitary, and peripheral glands.
The Most Widely Used and Accessible Systems
- Calcium indicators
- Voltage indicators
Calcium is a universal second messenger, playing an essential role in excitable cells and signal transduction. In neurons, action potentials (APs) trigger large and rapid calcium (Ca2+) influx through voltage-gated channels. AP firing can therefore be assessed by measuring changes in concentrations of intracellular Ca2+. Genetically encoded calcium indicators (GECIs) and small-molecule calcium-sensitive dyes such as fura-2 are both used to report changes in intracellular Ca2+ concentration; but GECIs have the advantage that they enable long-term, repeated non-invasive imaging of specific cells and compartments. State-of-the-art GECIs include Förster Resonance Energy Transfer (FRET)-based sensors, but for in vivo experiments particularly, the single-wavelength sensor GCaMP family has become the default tool. GCaMPs are based on circularly permuted green fluorescent protein (cpGFP), calmodulin (CaM), and the Ca2+/CaM-binding “M13” peptide. Ca2+ binding to the calmodulin domain results in a structural shift that enhances the fluorescence by deprotonation of the fluorophor.
Voltage imaging can directly display membrane potential dynamics and has been used to measure neural dynamics for decades. Genetically encoded voltage indicators (GEVIs) can be categorised into three classes based on their molecular structure and voltage sensing mechanism: voltage-sensitive domain-based sensors, rhodopsin-based sensors, and rhodopsin-FRET sensors for a comparative review on GEVIs.
Fig.1 Deep-brain imaging as a powerful tool to understand neuroendocrine functioning. (Campos, 2020)
- Viral vectors
Viruses can be used to target Ca2+ indicators to specific cells in vivo. Adeno-associated virus (AAV) and lentivirus (LV) are the most common viral vectors used today for neuroscience applications and result in robust expression of the desired gene throughout the cell. The viral approach has several advantages, including topographic specificity and the ability to control the amount of protein transduced.
To work with animals with similar levels of transgene expression within the targeted neuronal phenotype, transgenic lines have been generated. Essentially, two approaches exist. In the first type of transgenic line, GCaMP is expressed under the control of specific promoters. The second and more common approach is to generate transgenic animals bearing GCaMP expression in specific cell types, by crossing a Cre-driver with a floxed-GCaMP mouse.
Tools for Deep-brain Imaging
- GRIN Lenses
GRIN lenses resemble simple glass needles, but they are in fact complex lenses with a radially varying index of refraction, which causes an optical ray to follow a sinusoidal propagation path through the lens. GRIN lenses combine refraction at the end surfaces along with continuous refraction within the lens. GRIN lenses are the perfect tool to perform in vivo imaging in deep-brain regions, relaying signals to a point above the skull surface where image acquisition can take place.
Fig.2 GRIN lenses. (Miyamoto, 2016)
- Prism Probes
Prism-probes are composed of a prism fixed to the bottom of a cylindrical GRIN relay lens. The slanted surface of the prism is coated with metal to reflect by 90° the light from the microscope to excite GCaMP cells located along the imaging face of the prism probe. These probes have been used for multi-layer cortical imaging and could be suitable for populations of hypothalamic neurons that are spatially distributed along the z-axis.
Deep brain imaging can be a potential tool for obtaining data about the hypothalamic-pituitary system that has long been considered impossible to obtain. Creative Biolabs has a very strong technical force in the field of neuroscience research, focusing on deep-brain imaging in recent years. We can develop customized deep-brain imaging methods for customer’s neuroscience research projects to make the greatest breakthrough in your project. Contact us for more information.
- Campos, P.; et al. Diving into the brain: deep-brain imaging techniques in conscious animals. J Endocrinol. 2020, 246: R33-R50.
- Miyamoto, D.; Murayama, M. The fiber-optic imaging and manipulation of neural activity during animal behavior. Neurosci Res. 2016, 103: 1-9.