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Thirst driving and suppressing signals encoded by distinct neural populations in the brain.

Oka Y, Ye M, Zuker CS - Nature (2015)

Bottom Line: The light-induced response is highly specific for water, immediate and strictly locked to the laser stimulus.In contrast, activation of a second population of subfornical organ neurons, marked by expression of the vesicular GABA transporter VGAT, drastically suppresses drinking, even in water-craving thirsty animals.These results reveal an innate brain circuit that can turn an animal's water-drinking behaviour on and off, and probably functions as a centre for thirst control in the mammalian brain.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Biophysics, Columbia College of Physicians and Surgeons, Howard Hughes Medical Institute, Columbia University, New York, New York 10032, USA [2] Department of Neuroscience, Columbia College of Physicians and Surgeons, Howard Hughes Medical Institute, Columbia University, New York, New York 10032, USA.

ABSTRACT
Thirst is the basic instinct to drink water. Previously, it was shown that neurons in several circumventricular organs of the hypothalamus are activated by thirst-inducing conditions. Here we identify two distinct, genetically separable neural populations in the subfornical organ that trigger or suppress thirst. We show that optogenetic activation of subfornical organ excitatory neurons, marked by the expression of the transcription factor ETV-1, evokes intense drinking behaviour, and does so even in fully water-satiated animals. The light-induced response is highly specific for water, immediate and strictly locked to the laser stimulus. In contrast, activation of a second population of subfornical organ neurons, marked by expression of the vesicular GABA transporter VGAT, drastically suppresses drinking, even in water-craving thirsty animals. These results reveal an innate brain circuit that can turn an animal's water-drinking behaviour on and off, and probably functions as a centre for thirst control in the mammalian brain.

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Activation of excitatory neurons in the subfornical organs (SFO) triggers immediate drinking behavior(a) Water-deprivation activates CamKII/nNOS-positive neurons in the SFO. Robust Fos expression was induced in the SFO after water restriction for 48-hr. Shown are double immunolabeling for Fos and CamKII. Most Fos positive neurons co-expressed CamKII (95.9 ± 0.3%, n=3); also shown is the co-expression of CamKII with nNOS. These neurons are excitatory as they are marked by a VGlut2 trasgenic reporter 24 (Extatended Data Fig 2). (b) Whole-cell patch-clamp recording from SFO CamKII-positive neurons in acute hypothalamic slices demonstrating light-induced activation of the ChR2-expressing neurons. Shown are traces of a representative neuron subjected to 40 pulses of ChR2 excitation (20 Hz; 2 ms pulses); blue bars denote the time and duration of the light stimulus. Scale bars, 50 μm. (c) Photostimulation of CamKII-positive neurons in the SFO (trials 7–12; blue shading) triggered intense drinking; each black bar indicates an individual licking event. In the absence of light stimulation the same water-satiated animal exhibits very sparse events of drinking (trials 1–6). (d) Success of inducing drinking by photostimulation of the SFO. The Drinking Response (%) was calculated by determining the number of trials with >5 licks over the total number of trials; animals were tested for >10 trials each (see Methods for details). The panel shows animals infected with AAV-CamKIIa-ChR2-EYFP (n=10; red bar), and control mice infected with AAV-CamKIIa-GFP (n=4; black bar); white bars indicate the responses in the absence of photostimulation (Mann-Whitney test P < 0.0003). (e) Quantitation of the volume of water consumed within 15 min by 3 groups of animals: water-restricted for 48-hr, water-satiated, and water-satiated but photostimulated during the test; light (20 Hz) was delivered with a regime of 30 s ON and 30 s OFF for the entire 15 min session (n=4, Mann-Whitney test, P < 0.03 for water-satiated ± light). Values are means ± s.e.m.
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Figure 1: Activation of excitatory neurons in the subfornical organs (SFO) triggers immediate drinking behavior(a) Water-deprivation activates CamKII/nNOS-positive neurons in the SFO. Robust Fos expression was induced in the SFO after water restriction for 48-hr. Shown are double immunolabeling for Fos and CamKII. Most Fos positive neurons co-expressed CamKII (95.9 ± 0.3%, n=3); also shown is the co-expression of CamKII with nNOS. These neurons are excitatory as they are marked by a VGlut2 trasgenic reporter 24 (Extatended Data Fig 2). (b) Whole-cell patch-clamp recording from SFO CamKII-positive neurons in acute hypothalamic slices demonstrating light-induced activation of the ChR2-expressing neurons. Shown are traces of a representative neuron subjected to 40 pulses of ChR2 excitation (20 Hz; 2 ms pulses); blue bars denote the time and duration of the light stimulus. Scale bars, 50 μm. (c) Photostimulation of CamKII-positive neurons in the SFO (trials 7–12; blue shading) triggered intense drinking; each black bar indicates an individual licking event. In the absence of light stimulation the same water-satiated animal exhibits very sparse events of drinking (trials 1–6). (d) Success of inducing drinking by photostimulation of the SFO. The Drinking Response (%) was calculated by determining the number of trials with >5 licks over the total number of trials; animals were tested for >10 trials each (see Methods for details). The panel shows animals infected with AAV-CamKIIa-ChR2-EYFP (n=10; red bar), and control mice infected with AAV-CamKIIa-GFP (n=4; black bar); white bars indicate the responses in the absence of photostimulation (Mann-Whitney test P < 0.0003). (e) Quantitation of the volume of water consumed within 15 min by 3 groups of animals: water-restricted for 48-hr, water-satiated, and water-satiated but photostimulated during the test; light (20 Hz) was delivered with a regime of 30 s ON and 30 s OFF for the entire 15 min session (n=4, Mann-Whitney test, P < 0.03 for water-satiated ± light). Values are means ± s.e.m.

Mentions: The subfornical organ (SFO) is one of several CVO nuclei activated by thirst-inducing stimuli (e.g. water-deprivation) 1,9. This nucleus lacks the normal blood brain barrier, and has been proposed to function as an osmolality sensor in the brain 1,14,15. We reasoned that if we could identify a selective population of neurons in the SFO that respond to dehydration, they might provide a genetic handle to explore the neural control of thirst and water-drinking behavior. Using Fos as a marker for neuronal activation, we found that approximately 30% of the SFO neurons are strongly labeled with Fos after a 48-hr water restriction regime (no Fos expression was observed under water-satiated conditions, Extended Data Figure 1b). Notably, essentially all of the Fos-labeled cells co-expressed Ca2+/calmodulin-dependent kinase II (CamKII; Figure 1a upper panel), a known marker of excitatory neurons (see Extended Data Figure 2), as well as neuronal nitric oxide synthase (nNOS; Figure 1a lower panel). If these SFO neurons function as key cellular switches in the circuit that drives water consumption, then their activation should trigger water-drinking responses.


Thirst driving and suppressing signals encoded by distinct neural populations in the brain.

Oka Y, Ye M, Zuker CS - Nature (2015)

Activation of excitatory neurons in the subfornical organs (SFO) triggers immediate drinking behavior(a) Water-deprivation activates CamKII/nNOS-positive neurons in the SFO. Robust Fos expression was induced in the SFO after water restriction for 48-hr. Shown are double immunolabeling for Fos and CamKII. Most Fos positive neurons co-expressed CamKII (95.9 ± 0.3%, n=3); also shown is the co-expression of CamKII with nNOS. These neurons are excitatory as they are marked by a VGlut2 trasgenic reporter 24 (Extatended Data Fig 2). (b) Whole-cell patch-clamp recording from SFO CamKII-positive neurons in acute hypothalamic slices demonstrating light-induced activation of the ChR2-expressing neurons. Shown are traces of a representative neuron subjected to 40 pulses of ChR2 excitation (20 Hz; 2 ms pulses); blue bars denote the time and duration of the light stimulus. Scale bars, 50 μm. (c) Photostimulation of CamKII-positive neurons in the SFO (trials 7–12; blue shading) triggered intense drinking; each black bar indicates an individual licking event. In the absence of light stimulation the same water-satiated animal exhibits very sparse events of drinking (trials 1–6). (d) Success of inducing drinking by photostimulation of the SFO. The Drinking Response (%) was calculated by determining the number of trials with >5 licks over the total number of trials; animals were tested for >10 trials each (see Methods for details). The panel shows animals infected with AAV-CamKIIa-ChR2-EYFP (n=10; red bar), and control mice infected with AAV-CamKIIa-GFP (n=4; black bar); white bars indicate the responses in the absence of photostimulation (Mann-Whitney test P < 0.0003). (e) Quantitation of the volume of water consumed within 15 min by 3 groups of animals: water-restricted for 48-hr, water-satiated, and water-satiated but photostimulated during the test; light (20 Hz) was delivered with a regime of 30 s ON and 30 s OFF for the entire 15 min session (n=4, Mann-Whitney test, P < 0.03 for water-satiated ± light). Values are means ± s.e.m.
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Figure 1: Activation of excitatory neurons in the subfornical organs (SFO) triggers immediate drinking behavior(a) Water-deprivation activates CamKII/nNOS-positive neurons in the SFO. Robust Fos expression was induced in the SFO after water restriction for 48-hr. Shown are double immunolabeling for Fos and CamKII. Most Fos positive neurons co-expressed CamKII (95.9 ± 0.3%, n=3); also shown is the co-expression of CamKII with nNOS. These neurons are excitatory as they are marked by a VGlut2 trasgenic reporter 24 (Extatended Data Fig 2). (b) Whole-cell patch-clamp recording from SFO CamKII-positive neurons in acute hypothalamic slices demonstrating light-induced activation of the ChR2-expressing neurons. Shown are traces of a representative neuron subjected to 40 pulses of ChR2 excitation (20 Hz; 2 ms pulses); blue bars denote the time and duration of the light stimulus. Scale bars, 50 μm. (c) Photostimulation of CamKII-positive neurons in the SFO (trials 7–12; blue shading) triggered intense drinking; each black bar indicates an individual licking event. In the absence of light stimulation the same water-satiated animal exhibits very sparse events of drinking (trials 1–6). (d) Success of inducing drinking by photostimulation of the SFO. The Drinking Response (%) was calculated by determining the number of trials with >5 licks over the total number of trials; animals were tested for >10 trials each (see Methods for details). The panel shows animals infected with AAV-CamKIIa-ChR2-EYFP (n=10; red bar), and control mice infected with AAV-CamKIIa-GFP (n=4; black bar); white bars indicate the responses in the absence of photostimulation (Mann-Whitney test P < 0.0003). (e) Quantitation of the volume of water consumed within 15 min by 3 groups of animals: water-restricted for 48-hr, water-satiated, and water-satiated but photostimulated during the test; light (20 Hz) was delivered with a regime of 30 s ON and 30 s OFF for the entire 15 min session (n=4, Mann-Whitney test, P < 0.03 for water-satiated ± light). Values are means ± s.e.m.
Mentions: The subfornical organ (SFO) is one of several CVO nuclei activated by thirst-inducing stimuli (e.g. water-deprivation) 1,9. This nucleus lacks the normal blood brain barrier, and has been proposed to function as an osmolality sensor in the brain 1,14,15. We reasoned that if we could identify a selective population of neurons in the SFO that respond to dehydration, they might provide a genetic handle to explore the neural control of thirst and water-drinking behavior. Using Fos as a marker for neuronal activation, we found that approximately 30% of the SFO neurons are strongly labeled with Fos after a 48-hr water restriction regime (no Fos expression was observed under water-satiated conditions, Extended Data Figure 1b). Notably, essentially all of the Fos-labeled cells co-expressed Ca2+/calmodulin-dependent kinase II (CamKII; Figure 1a upper panel), a known marker of excitatory neurons (see Extended Data Figure 2), as well as neuronal nitric oxide synthase (nNOS; Figure 1a lower panel). If these SFO neurons function as key cellular switches in the circuit that drives water consumption, then their activation should trigger water-drinking responses.

Bottom Line: The light-induced response is highly specific for water, immediate and strictly locked to the laser stimulus.In contrast, activation of a second population of subfornical organ neurons, marked by expression of the vesicular GABA transporter VGAT, drastically suppresses drinking, even in water-craving thirsty animals.These results reveal an innate brain circuit that can turn an animal's water-drinking behaviour on and off, and probably functions as a centre for thirst control in the mammalian brain.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Biophysics, Columbia College of Physicians and Surgeons, Howard Hughes Medical Institute, Columbia University, New York, New York 10032, USA [2] Department of Neuroscience, Columbia College of Physicians and Surgeons, Howard Hughes Medical Institute, Columbia University, New York, New York 10032, USA.

ABSTRACT
Thirst is the basic instinct to drink water. Previously, it was shown that neurons in several circumventricular organs of the hypothalamus are activated by thirst-inducing conditions. Here we identify two distinct, genetically separable neural populations in the subfornical organ that trigger or suppress thirst. We show that optogenetic activation of subfornical organ excitatory neurons, marked by the expression of the transcription factor ETV-1, evokes intense drinking behaviour, and does so even in fully water-satiated animals. The light-induced response is highly specific for water, immediate and strictly locked to the laser stimulus. In contrast, activation of a second population of subfornical organ neurons, marked by expression of the vesicular GABA transporter VGAT, drastically suppresses drinking, even in water-craving thirsty animals. These results reveal an innate brain circuit that can turn an animal's water-drinking behaviour on and off, and probably functions as a centre for thirst control in the mammalian brain.

Show MeSH
Related in: MedlinePlus