Project 1

In this series of studies funded by the NIH, we will use a unique genetics-based approach to identify molecules in specialized brainstem cells that enable them to detect changes in carbon dioxide (CO2) or pH. Understanding these fundamental neural mechanisms is critical to complete the understanding of respiratory-related diseases thought to result from altered chemoreception in humans, including sudden infant death syndrome (SIDS). 

How can we use a genetics-based approach to finding pH sensors in cells? The concept is based on the idea that we can use a cell sorter to sort out the cells we want to study, and then measure every gene they express and how much of each is there. For example, we know that not all neurons that produce serotonin are pH sensitive. So, if we can measure which genes are expressed in the pH sensitive ones which are absent or in lower abundance in the pH insensitive ones, we might get an idea of the genetic determinants of cellular pH sensitivity. 

We can either give high CO2 or low pH solutions to cells in a dish (acute brainstem slices) or expose animals like rats to normal air or and environment enriched with CO2 (hypercapnia). This will increase the activity of pH pr CO2 sensitive cells and increased their production of a protein called c-fos. Then, we can use a cell sorter to sort out the 5-HT neurons that express GFP (Fig. 1) and further sort out cells that are c-fos positive using an antibody that recognizes this protein (green dots in Fig. 2). So, we can then extract messenger RNA (mRNA) from the pools of sorted cells, and compare pH sensitive 5-HT neurons (c-fos+ and GFP+) and pH insensitive 5-HT neurons (c-fos- and GFP+) with RNA sequencing. 

 

Using a similar approach we identified two pH sensitive potassium ion channel genes that are upregulated with age in 5-HT neuron-enriched cell pools - kcnj10 (Kir4.1) and kcnj16 (kir5.1). Using immunoflourescence staining we found that Kir4.1 was only found to be expressed in nearby glial cells and not the 5-HT neurons. However, we did find that Kir5.1 is expressed in 5-HT neurons (Fig. 3), along with other neurons and glial cells. Could Kir5.1 contribute to pH sensitivity in 5-HT neurons? 

Once a candidate pH sensing ion channel is identified (like Kir5.1), we will use patch clamp recordings of individual GFP 5-HT neurons to see which are pH sensitive, and if so if we can block that response with a drug that blocks the ion channel of interest (Fig. 4). We can also validate the gene as being important for pH homeostasis by generating mutations in the gene to created knockout rats. These studies are ongoing.

Fig. 4 - Strategy for validation of pH sensing ion channel candidates

Fig. 1 - Green fluorescence (GFP) is restricted to 5-HT producing neurons in transgenic SS-T2ePet-eGFP rats (Katter et al., 2013).

Fig. 2 - Immunoflourescence images of medullary raphe 5-HT neurons (red) from rats under control (room air exposed) or hypercapnic (7% CO2) conditions. Note that CO2 exposure increases c-fos (green dots) expression, indicating some 5-HT neurons are activated by CO2. We seek to understand if this actvation is intrinsic to these cells through pH or CO2 sensitive ion channel activity.

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Fig. 3 - Strategy for validation of pH sensing ion channel candiates