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Sodium regulates clock time and output via an excitatory GABAergic pathway

Nature volume 583, pages 421–424 (2020)Cite this article

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Abstract

The suprachiasmatic nucleus (SCN) serves as the body’s master circadian clock that adaptively coordinates changes in physiology and behaviour in anticipation of changing requirements throughout the 24-h day–night cycle1,2,3,4. For example, the SCN opposes overnight adipsia by driving water intake before sleep5,6, and by driving the secretion of anti-diuretic hormone7,8 and lowering body temperature9,10 to reduce water loss during sleep11. These responses can also be driven by central osmo-sodium sensors to oppose an unscheduled rise in osmolality during the active phase12,13,14,15,16. However, it is unknown whether osmo-sodium sensors require clock-output networks to drive homeostatic responses. Here we show that a systemic salt injection (hypertonic saline) given at Zeitgeber time 19—a time at which SCNVP (vasopressin) neurons are inactive—excited SCNVP neurons and decreased non-shivering thermogenesis (NST) and body temperature. The effects of hypertonic saline on NST and body temperature were prevented by chemogenetic inhibition of SCNVP neurons and mimicked by optogenetic stimulation of SCNVP neurons in vivo. Combined anatomical and electrophysiological experiments revealed that osmo-sodium-sensing organum vasculosum lamina terminalis (OVLT) neurons expressing glutamic acid decarboxylase (OVLTGAD) relay this information to SCNVP neurons via an excitatory effect of γ-aminobutyric acid (GABA). Optogenetic activation of OVLTGAD neuron axon terminals excited SCNVP neurons in vitro and mimicked the effects of hypertonic saline on NST and body temperature in vivo. Furthermore, chemogenetic inhibition of OVLTGAD neurons blunted the effects of systemic hypertonic saline on NST and body temperature. Finally, we show that hypertonic saline significantly phase-advanced the circadian locomotor activity onset of mice. This effect was mimicked by optogenetic activation of the OVLTGAD→ SCNVP pathway and was prevented by chemogenetic inhibition of OVLTGAD neurons. Collectively, our findings provide demonstration that clock time can be regulated by non-photic physiologically relevant cues, and that such cues can drive unscheduled homeostatic responses via clock-output networks.

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Fig. 1: HS reduces Tb and TBAT via SCNVP neurons.
Fig. 2: Osmo-sodium-sensitive OVLTGAD neurons excite SCNVP neurons at ZT19.
Fig. 3: OVLTGAD neurons mediate the effects of systemic HS on Tb and TBAT.
Fig. 4: Activation of the OVLTGAD→SCNVP pathway advances the onset of locomotor activity.

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Acknowledgements

This work was supported by a Foundation Grant from the Canadian Institutes of Health Research (CIHR; FDN 143337) and a James McGill Chair to C.W.B., and a CIHR Banting and Best Canada Graduate Scholarship awarded to C.G. We thank N. Cermakian, Z. Knight and F. Storch for valuable comments on an early draft of this manuscript; N. J. Simpson for technical assistance with some of the histological procedures; and H. Gainer (NIH) for providing the PS-41 monoclonal anti-AVP-neurophysin antibody. The RIMUHC receives generous funding from the Fonds de Recherche Québec Santé.

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Authors and Affiliations

  1. Brain Repair and Integrative Neuroscience Program, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada

    Claire Gizowski & Charles W. Bourque

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  1. Claire Gizowski
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  2. Charles W. Bourque
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Contributions

C.G. and C.W.B. designed the study, interpreted the results and wrote the manuscript. C.G. performed all of the experiments.

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Correspondence to Claire Gizowski or Charles W. Bourque.

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The authors declare no competing interests.

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Peer review information Nature thanks Chris Colwell, Michael Joseph McKinley and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 HS-induced decreases in Tb and TBAT can be measured using IRT and are not mediated by reduced locomotor activity.

a, The schematic illustrates a mouse with a subcutaneously implanted NanoTag monitoring device, which is used to track temperature via embedded thermocouple (TC). The mouse was simultaneously filmed from above using a thermography camera and eye temperature (TEYE) was assessed offline using FLIR software. b, Plots show changes in TC and TEYE recorded simultaneously in a mouse injected with NaCl (s.c.) to provoke a lowering of body temperature. Changes in TEYE and TC parallel each other and TEYE is about 1 °C higher than TC. c, Graph shows all TC samples plotted as a function of TEYE. There is a strong correlation between values obtained with the two methods. d, Plots show the mean ± s.e.m. average locomotor activity measured 10 min after s.c. injection of saline or 2 M NaCl in wild-type C57BL/6 mice (saline n = 8, NaCl n = 8; two-sided t-test).

Source data

Extended Data Fig. 2 Chemogenetic inhibition of SCNVP neurons.

a, AVPCre mice received bilateral injections of AAV DIO-hM4D(Gi) into the SCN and were allowed to recover for >3 weeks. b, Intense expression of the mCherry reporter could be seen in neurons of the rostral, mid and caudal SCN. The bottom panel reveals the nucleus boundaries by DAPI stain. c, d, Our injection strategy could mediate mCherry expression in SCNVP neurons in b, without affecting VP neurons in the supraoptic nucleus (SON; c) or the paraventricular nucleus (PVN; d). Similar results were seen in two mice.

Extended Data Fig. 3 Optogenetic activation of SCNVP neurons.

a, AVPCre × ChETAFlex mice were implanted with a fibreoptic (FO) allowing optogenetic excitation of SCNVP neurons using BL (473 nm). These mice were implanted in the midline, through the third ventricle (3V), with a FO probe targeting the dorsal edge of the SCN (the top image is a photograph illustrating the arrangement shown as a schematic in the bottom panel). The photograph shows that BL diffuses bilaterally to both SCNs. The FO probe was mounted through a magnetic coupler, which allowed attachment of the patch cord carrying BL from the 473-nm laser. b, The schematic illustrates the histologically determined position of the FO tip for the mice used in this study. c, Bar graph shows mean (±s.e.m.) changes in locomotor activity induced by BL (+BL; 5 Hz, 50-ms pulse, 23 mW, 30 min) delivered to the SCNs of AVPCre × ChETAFlex mice instrumented as shown in a. Stimulation of SCNVP neurons with +BL did not significantly affect locomotor activity at 30 min compared to baseline (average locomotor activity measured before optogenetic stimulation; n = 6, mean ± s.e.m., two-sided paired t-test).

Source data

Extended Data Fig. 4 Specific optogenetic activation of SCNVP neurons mimics the effects of HS on Tb and TBAT.

a, AVPCre mice received bilateral injections of AAV DIO-ChR2-EYFP into the SCN and were allowed to recover for >3 weeks. b, EYFP expression in the SCN of a live slice (left). Boundaries of the SCN by DAPI stain in the same slice but post-fix (right). Similar results were seen in n = 4. c, Graph shows mean ± s.e.m. ΔTb after 30 min of optogenetic stimulation of AVPCre neurons with +BL (5 Hz, 30-ms pulse, 8–13 mW, 30 min; n = 4; paired t-test). d, Graph shows mean ± s.e.m. ΔTb of a time control with no blue light (−BL; n = 3, Wilcoxon signed-rank test). e, Graph shows mean ± s.e.m. ΔTBAT after 30 min of optogenetic stimulation with +BL (5 Hz, 30-ms pulse, 8–13 W; n = 4; paired t-test). f, Graph shows mean ± s.e.m. ΔTBAT after 30 min of time control −BL (n = 3; paired t-test). All tests are two-sided.

Source data

Extended Data Fig. 5 OVLT→SCN neurons are osmo-sodium sensitive.

a, Schematic of a unilateral injection of retrograde microspheres in the SCN of a wild-type C57BL/6 mouse. b, Merged brightfield and fluorescence micrograph showing the site of injection (beads in green) in one of four mice tested. c, Confocal image of a coronal OVLT section from the brain in b. The pink arrows point to OVLT neurons labelled with retrogradely transported beads. The dashed line delineates the OVLT. d, Mice were injected s.c. with 2 M NaCl and perfused with fixative 90 min later. Expression of FOS was observed in retrogradely labelled OVLT neurons. Similar results were observed in n = 4. e, Wild-type mice were injected s.c. with 2 M NaCl and perfused with fixative 90 min later. The micrograph shows the expression of immunolabelled FOS in red and GAD67, a marker for GABA neurons, in green in the OVLT. The yellow arrows point to FOS+/GAD67− neurons, and the white arrows point to FOS+/GAD67+ neurons (n = 1).

Extended Data Fig. 6 OVLTGAD fibres terminate within the SCN.

a, GADCre mice were injected with AAV-DIO-HM4D(Gi)-mCherry into the OVLT. b, Mice showed intense expression of the mCherry reporter in OVLTGAD neurons. c, The boundaries of the SCN were revealed by DAPI stain (dashed white circle). d, The white arrows point to mCherry-labelled OVLTGAD axons within the same SCN section in c. Similar results were observed in n = 4.

Extended Data Fig. 7 GABAAR antagonists reduced spontaneous action potential firing rate of SCNVP neurons.

a, Frequency plot shows changes in firing rate evoked by 20 μm GBZ on an SCNVP neuron in the presence of 3 mM Kyn. b, Bar graph shows mean ± s.e.m. firing rate observed in the absence (control) and presence of GABAAR antagonists (Bic or GBZ), in seven neurons from five slices (two-sided paired t-test).

Source data

Extended Data Fig. 8 Optogenetic stimulation of ChR2-expressing OVLTGABA neurons.

a, Cell-attached recording of an OVLTGABA neuron expressing ChR2 driven by local delivery of AAV fDIO-ChR2-EYFP in a VGATFlp × AVPCre mouse. Note the spiking response to every light pulse. b, Plots of efficacy of optogenetic excitation (per cent activation) measured in five neurons from two slices. All cells display 100% excitation when exposed to blue light with an intensity of >4 mW. c, Repetitive optogenetic stimulation at 2.5 Hz mediated sustained excitation for 20 min (only 10 min is shown).

Source data

Extended Data Fig. 9 Chemogenetic inhibition of OVLTGAD neurons.

a, Schematic illustrating a coronal view of the brain at the level of the OVLT, median preoptic nucleus (MnPO) and anterior commissure (AC). GADCre mice were injected with AAV-DIO-hM4D(Gi) into the OVLT and were allowed to recover for >3 weeks. b, Mice showed intense expression of the mCherry reporter. c, Histological assessment of the location of AAV-driven expression based on inspection of the mCherry signal in the eight mice used in this study. All injections included the OVLT.

Extended Data Fig. 10 Optogenetic activation of GABAergic OVLT neuron axon terminals in the SCN.

a, GADCre mice were injected with AAV DIO-ChR2-EYFP into the OVLT and were allowed to recover for >3 weeks. b, Mice showed intense expression of the EYFP reporter in the OVLT area. c, Histological assessment of the location of AAV-driven expression based on inspection of the EYFP signal in the five mice used in this study. All injections included the OVLT. d, Relative placement of the FO probes in the same five mice. e, Stimulation of OVLTGAD neuron axon terminals with +BL (2.5 Hz, 20-ms pulse, 5–10 mW, 30 min) did not significantly affect the average locomotor activity at 30 min compared to baseline (the average locomotor activity measured before optogenetic stimulation; n = 5, mean ± s.e.m., two-sided paired t-test).

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Gizowski, C., Bourque, C.W. Sodium regulates clock time and output via an excitatory GABAergic pathway. Nature 583, 421–424 (2020). https://doi.org/10.1038/s41586-020-2471-x

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