Measuring neuronal activity in response to gut signalling

The understanding of gut signalling effects on neuronal activity has been studied in both humans and animal models. In humans, this effect has been explored through the use of fMRI ^{61, 62}. In animals, the study of in vivo neural dynamics in the context of feeding behaviour became possible with recent advances in transgenic models, neurotransmitter sensing, and development imaging techniques with high temporal and spatial resolution ⁶³.

Currently, it is possible to monitor in vivo neurotransmitter dynamics and neuronal activity responses.

The neurotransmitter that has been studied the most in the context of feeding behaviour is dopamine.

Several works have reported the use of microdialysis ²⁷ and cyclic voltammetry ³⁵ to measure dopamine release while animals were exposed to caloric and non-caloric solutions. By measuring neurotransmitter dynamics these techniques are an indirect measure of neuronal activity. Microdialysis is performed in awake freely behaving animals and it is based on sampling of molecules from interstitial space that allows quantification of neurotransmitters, such as dopamine ^{63, 64}. Nevertheless, it has slow temporal dynamics ⁶³. On the other hand, cyclic voltammetry can be performed on anesthetized or awake animals and consists of measuring the current response of a redox solution with a linearly cycled potential sweep. Therefore, it involves applying voltage to induce oxidation and reduction of a chemical that, in this context, is a neurotransmitter ^{63, 65}. It has the advantage of having a higher temporal resolution, when compared to microdialysis but since it is an electrochemical technique, it is unspecific ⁶⁵. One disadvantage that these techniques presented was the inability to monitor and measure long-term changes which is crucial to understand how neurotransmitters can modulate feeding behaviour over time ⁶³.

More recently, fibre photometry using dopamine sensors have been used. The development of dopamine sensors with high spatial and temporal resolution permitted the usage of this technique for measurements of dopamine dynamics in the striatum (Figure 1.4). This approach involves the implant of, at least, one small optical fibre that allows to monitor population-level dynamics. For most cases, fibre photometry relays on calcium indicators to measure overall calcium dynamic in neuronal population ⁶⁶. Additionally, this technique can also be used along with dopamine indicators in order to exclusively monitor and record dopamine

dynamics in awake freely behaving mice ⁶⁷. This indicator is sensitive to dopamine concentration changes because its conformation changes upon dopamine binding. Also, it was engineered to be couples to a green fluorescent protein (GFP) and, thus, when dopamine binds to this indicator it induces changes in fluorescence so the more dopamine binds, the more intense the fluorescence ⁶⁷.

Calcium imaging, contrary to all the techniques mentioned above, allow to directly monitor and quantify neuronal activity of individual neurons (Figure 1.4). It relays on calcium indicators to measure activity, but the spatial resolution provided allows to monitor fluorescence changes of individual neurons and, consequently, identify neuronal subpopulations. Calcium indicators are proteins that change conformation upon calcium binding and, subsequently, emit fluorescence. These are a good proxy for neuronal activity since calcium floods neurons when action potentials are generated and, if the cell expresses a calcium indicator, fluorescence increases in this scenario. ⁶⁶ Currently, the most common calcium indicator is green calmodulin protein – GCaMP. Fibre photometry is relatively non-invasive and has the advantage of allowing to measure neuronal dynamic in freely behaving animals ⁶³.

Additionally, this approach also allows to image calcium dynamics of two cell populations by using calcium indicators that emit different colours. Calcium imaging involves the implant of a gradient index refractive (GRIN) lens and the use of a small head mounted microscope (miniscope) ⁶⁸. It is less invasive than, for example, 2-photon microscopy and allows to easily monitor neuronal activity in awake freely behaving animals ⁶³.

Most of the techniques described above take advantage of transgenic animal models in order to monitor activity of a specific neuronal population. These models allow to express calcium indicators in a specific cell type, for example, dopaminergic neurons by using the Cre-loxP system ⁶⁹. The Cre recombinase recognizes two consecutive loxP sites and enable the expression of the calcium indicator in cells that express the enzyme Figure 1.4 Optical imaging techniques for awake freely behaving animals.

Schematic representation of fibre photometry (left) and calcium imaging (right) experiments. In fibre photometry experiments mice undergo surgeries that allow to observe calcium dynamics with the help of optical fibres. For calcium imaging experiments, a GRIN lens and a miniscope is used to monitor neuronal activity. Fibre photometry is a has less temporal resolution and does not enable identification of subpopulation of neurons, therefore the spatial resolution is limited when compared to calcium imaging.

Cre recombinase ^{69, 70}. Therefore, Cre transgenic mice are genetically modified animals that express Cre recombinase under a cell specific transporter.

These techniques along with nutrient infusions, FNC assays, and sham-feeding experiments have allowed to better understand how gut signalling influences neuronal activity. The constant development and optimization of brain imaging techniques has granted the possibility to image deep brain regions which was not possible until very recently ⁶⁸. At the same time, neuronal imaging has provided the opportunity to ask new questions and better understand the neuronal underpinnings behind food-seeking behaviour.