A gut-to-brain signal of fluid osmolarity controls thirst satiation

Welcome to our Monthly Journal Club! Each month I post a paper or two that I have read and find interesting. I use this as a forum for open discussion about the paper in question. Anyone can participate in the journal club, and provide comments/critiques on the paper by leaving a comment below. I picked this months paper because it reveals a circuit spanning multiple systems and timescales influencing one of our most essential behaviors, drinking and fluid intake. This month’s paper is “A gut-to-brain signal of fluid osmolarity controls thirst satiation” by Zachary Knight and colleagues at The University of California - San Francisco. The lead author was Christopher A. Zimmerman, a graduate student in Dr. Knight’s lab who focuses on homeostatic control of thirst. I will provide a brief overview of the techniques/approaches used to make it more understandable to non-expert readers. If I can’t figure something out, I’ll just say so.

How do we know when we’re thirsty, and how do we know when to stop drinking? This is very important, as we need to keep proper concentrations of ions (also known as osmolarity) within the fluid compartments of our body to stay alive. Osmolarity can be thought of as the relative concentrations of ions in a solution, where our body likes to stay around 300 milli-osmoles (mOsm), where each compartment (intra-cellular, interstitial, and blood) remain in equilibrium. If you drink too much water, you can die due to the drastic changes in osmolarity that occur, causing our cells to burst (lysis). If you are dehydrated, our cells wrinkle up (crenation), a phenomenon that can also lead to death. A jarring example of this occurred in 2007 when a radio station held a contest titled “Hold your Wee for a Wii”, where contestants were tasked with drinking as much water as they could without peeing to win a Nintendo Wii. Unfortunately, the radio DJs did not know anything about basic physiology and were under the false impression that you can drink as much water as you want without any detrimental effects. One contestant drank so much that the osmolarity of her blood became drastically different from other fluid compartments in her body leading to cell lysis, and she died as a result.

There are several well-known circuits in the brain that have been identified as key regulators of thirst and drinking behavior. These include the subfornical organ (SFO), the median preoptic nuclei (MnPO), and the supraoptic nuclei (SON). Together, these structures receive input from the mouth and throat on the amount of liquid that we are drinking in real time (that is, they detect the volume of fluid intake), and are rapidly inactivated upon drinking pretty much any type of fluid (See the Figure below). After not drinking for a while (i.e., we’re thirsty), the activity in these structures rises and promotes drinking behavior. There’s something that doesn’t quite add up here, and that is how do these structures know the ‘type’ of fluid that you’re drinking? If it is just measuring the volume, then we’d feel just as quenched after drinking a bottle of sea water as we would after drinking one of fresh water. The only real difference between fresh and sea water is the salt content (that is, sea water has a higher osmolarity than fresh water). That means that there must be an osmolarity detector somewhere in the body that relays to the brain information about the type of fluid that has been consumed. As Dr. Knight says, "There has to be a mechanism for the brain to track how salty the solutions that you drink are and use that to fine-tune thirst…But the mechanism was unknown."

Brain structures underlying thirst, drinking, and satiation. A major component is the subfornical organ (SFO), which receives input on the volume of fluid consumed, and directs changes in drinking behavior using excitatory (glutamate) and inhibitory (GABA) signaling. (Credit:    Zimmerman et al., 2017   )

Brain structures underlying thirst, drinking, and satiation. A major component is the subfornical organ (SFO), which receives input on the volume of fluid consumed, and directs changes in drinking behavior using excitatory (glutamate) and inhibitory (GABA) signaling. (Credit: Zimmerman et al., 2017)

Christopher Zimmerman and other members of the team tested this using a method called fiber photometry in tandem with intra-gastric (i.e., into the gut) injections of fluids with different osmolarity. Fiber photometry is a way to measure the activity of dozens or hundreds of cells in deep brain structures simultaneously and in real time, while an animal (in this case, a mouse) behaves and runs around normally. This makes it a great tool to see how different populations of neurons operate in real-world scenarios. Fiber photometry allowed the authors to see how neurons in the subfornical organ (SFO) respond when a thirsty mouse drinks naturally (in hydrated and dehydrated conditions), and how they respond when liquids of different osmolarity are injected directly into the gut (allowing them to bypass the volume sensors in the mouth). They confirmed prior studies that showed rapid reductions in SFO activity upon drinking either regular water or salty water. However, when they injected these liquids into the gut, the SFO reduced its activity only in conditions where normal water was injected, but not in response to salt water. This suggests that a signal from the gut makes it up to the brain, where it somehow conveys to the brain the osmolarity of a liquid that has been consumed (see the figure below)

The subfornical organ (SFO) rapidly reduces activity upon drinking either regular (Water) or salty (NaCl) water (panel b). However, when solutions of different salt concentrations (osmolarity) were directly infused into the gut (panel d), the SFO drastically increased its activity (panels e,f,g) as a function of the osmolarity of the solution (R^2 = 0.98; also known as a near 1 to 1 relationship). (note: F/F means ‘fractional fluorescent change’, indicating the activity of the cells being measured) (Credit: Zimmerman et al., 2019).

The subfornical organ (SFO) rapidly reduces activity upon drinking either regular (Water) or salty (NaCl) water (panel b). However, when solutions of different salt concentrations (osmolarity) were directly infused into the gut (panel d), the SFO drastically increased its activity (panels e,f,g) as a function of the osmolarity of the solution (R^2 = 0.98; also known as a near 1 to 1 relationship). (note: F/F means ‘fractional fluorescent change’, indicating the activity of the cells being measured) (Credit: Zimmerman et al., 2019).

The authors moved on to investigate how the osmolarity signal is represented in other components of the thirst circuit (i.e., MnPO, SON). Targeting vasopressin neurons in the SON, they showed that these neurons act in a similar fashion to those in the SFO. They are rapidly inhibited by drinking, but also increase activity in response to elevations in blood osmolarity (see Figure below).

Supraoptic nucleus (SON) vasopressin neurons are rapidly inactivated by drinking (panel C), and bidirectionally regulated by gut fluid osmolarity (panel d). The heat maps in (d) show the activity of these neurons where warmer colors represent higher activity. Note that increases in fluid salt content (150 mM to 500 mM) causes stepwise increases in neural activity. (Credit: Zimmerman et al., 2019).

Supraoptic nucleus (SON) vasopressin neurons are rapidly inactivated by drinking (panel C), and bidirectionally regulated by gut fluid osmolarity (panel d). The heat maps in (d) show the activity of these neurons where warmer colors represent higher activity. Note that increases in fluid salt content (150 mM to 500 mM) causes stepwise increases in neural activity. (Credit: Zimmerman et al., 2019).

Continuing their investigation of neural circuitry controlling drinking behavior and fluid balance, they measured how the final major component in the circuit (the MnPO) alters activity in different experimental paradigms. This time, they upgraded their tech from fiber photometry to using a tiny microscope (microendoscope) implanted into the mouse’s brain to see how the individual cells in the MnPO respond to fluid intake and blood osmolarity. The picture below shows the similarities and differences of microendoscopy and fiber photometry.

Both  in vivo  microendoscopy (a) and fiber photometry (b) measure neural activity by collecting light emitted by a fluorescent genetically encoded calcium indicator in neurons of interest (e.g., GCaMP6s). More cumbersome and harder to use, the microendoscope technique allows researchers to examine the activity of individual cells over long periods of time in awake, behaving mice. This is a major advantage over fiber photometry, as it allows one to understand how different cells in the circuit act in response to various stimuli. (Credit:    Resendez & Stuber, 2015   ).

Both in vivo microendoscopy (a) and fiber photometry (b) measure neural activity by collecting light emitted by a fluorescent genetically encoded calcium indicator in neurons of interest (e.g., GCaMP6s). More cumbersome and harder to use, the microendoscope technique allows researchers to examine the activity of individual cells over long periods of time in awake, behaving mice. This is a major advantage over fiber photometry, as it allows one to understand how different cells in the circuit act in response to various stimuli. (Credit: Resendez & Stuber, 2015).

Using this technique, they started by targeting neurons that make glutamate (excitatory) in the MnPO. They observed that individual neurons in this region could be clustered into three categories based on how they responded to changes in fluid intake and osmolarity. One subpopulation (cluster 1, 17%) didn’t show any response to regular saline injection but showed significant activation after salt challenge, suggesting that these neurons encode blood osmolarity. These same neurons were drastically inhibited during drinking. By contrast, neurons that fell into cluster 2 (34%) showed only quick responses independent from fluid intake (the authors think it was probably the stress or pain of injection), whereas neurons from cluster 3 (49%) were largely unresponsive.

They continued to investigate another major neural population in this region using the same technique, those that express the inhibitory neurotransmitter GABA. They also were able to segregate cells into different clusters based on their responses to fluid intake and osmolarity. Three different categories emerged. Specifically, they showed that individual neurons can be categorized as “ingestion-activated” (28%), “ingestion-inhibited” (36%), or “untuned” (35%). As the category names suggest, this means that different subsets of cells respond to fluid intake by increasing their activity, decreasing their activity, or not changing their activity at all (untuned; see panel [d] in the figure below).

GABA-producing neurons within the median preoptic nucleus (MnPO) can be clustered into “ingestion-activated”, “ingestion-inhibited”, or “untuned” based on their responses to fluid intake. in panel (d) we can clearly see segregation of these neural responses following drinking. (Credit: Zimmerman et al., 2019)

GABA-producing neurons within the median preoptic nucleus (MnPO) can be clustered into “ingestion-activated”, “ingestion-inhibited”, or “untuned” based on their responses to fluid intake. in panel (d) we can clearly see segregation of these neural responses following drinking. (Credit: Zimmerman et al., 2019)

This demonstrates that individual MnPO glutamatergic neurons receive ingestion signals from the mouth/throat, satiation signals from the gut and homeostatic signals from the blood, which they process and integrate to estimate physiological state. Additionally, these data also indicate that the majority of GABAergic MnPO neurons are strongly influenced by fluid ingestion, with smaller subsets that integrate multiple signals with relevance to fluid balance (like water availability, stress and gastrointestinal osmolarity). Importantly, these studies suggest that the concept of homeostatic need (or physiological set point) can be computed at the level of individual neurons in a circuit. These findings could point the way for new therapies for diseases that drastically alter fluid balance in the body, such as diabetes and cardiovascular disease. Additional studies on regulation of homeostasis are required to understand how these populations of cells act together to receive, integrate, and relay a signal that engages drinking behavior and the feeling of ‘thirst’.

As always, let me know what you think by leaving a comment below or messaging me on twitter @jborniger ! See you guys next time! Stay curious!