05 Mar 2024

During sleep, the brain cleans itself, turning up the flow of cerebrospinal fluid through the gray matter parenchyma to wash away waste. What powers this flow? In the February 28 Nature online, researchers led by Jonathan Kipnis at Washington University in St. Louis identified neuronal activity as the driving force. In unconscious mice, waves of ions moved through the parenchyma in sync with the electrical rhythms of delta slow-wave sleep. When the authors silenced neuronal activity, these waves ebbed, and solutes moved only sluggishly through the brain. Boosting neuronal activity did the opposite, amplifying ionic waves and speeding the passage of solutes.

Perhaps the purpose of synchronized neuronal firing during sleep is to create these ion waves to rinse away the day’s metabolic waste, the authors speculated. “Neurons that fire together, ‘shower’ together,” Kipnis and colleagues quipped.

A second paper in the February 28 Nature broadens evidence that neuronal activity can promote CSF flow. Researchers led by Li-Huei Tsai at the Massachusetts Institute of Technology, Cambridge, linked much faster 40 Hz gamma brain rhythms to more vigorous arterial constriction and dilation. This pulsation pushes solutes through the brain, dumping them out to cervical lymph nodes. The finding may help explain how stimulating awake animals with light and sound at 40 Hz clears amyloid, they noted (Mar 2019 news; May 2019 news).

“[These papers] provide direct evidence that neuronal activation can accelerate glymphatic waste clearance from the brain,” Maiken Nedergaard and Lauren Hablitz at the University of Rochester Medical Center, New York, wrote in an accompanying News & Views. Nedergaard first proposed the concept of glymphatic clearance. According to it, CSF arrives alongside penetrating arteries, washes through the parenchyma as interstitial fluid (ISF) aided by glia, and, freshly loaded with waste solutes, exits the brain along its veins (Aug 2012 news; Mar 2013 news).

Some previous work had hinted at a connection between neuronal firing and glymphatic flow. Hablitz and Nedergaard reported that the stronger the delta EEG signal was in unconscious mice, the faster CSF moved through their brain (Hablitz et al., 2019). Meanwhile, Laura Lewis at Boston University found that, in sleeping people, CSF passed through the brain in waves that were synchronized with slow-wave sleep (Nov 2019 news). However, such correlations do not prove that sleep waves power flow, and the hypothesis remained controversial (Hladky and Barrand, 2022). 

Seeking mechanistic evidence, Kipnis and colleagues turned to mice, in which chemogenetic and optogenetic tools enabled them to activate and silence neurons at will (Nov 2012 series). First author Li-Feng Jiang-Xie used ketamine to put mice into an unconscious state that mimics sleep, with neurons firing in slow 0.5 to 4 Hz delta waves. Because action potentials activate sodium/potassium pumps in the neuronal membrane, this synchronized firing created waves of ions. To detect them, Jiang-Xie and colleagues inserted a probe into the hippocampus; this picked up the electrical field in the ISF, which they reasoned could serve as a proxy measure of ion flux.

“This approach is years ahead of its time, because researchers are not yet able to visualize interstitial fluid or ion flow in the brain in real time,” Hablitz and Nedergaard noted.

What did Jiang-Xie and colleagues find? Ionic ISF waves were linked in time to delta sleep waves—as Lewis had described in people using different methodology.

Next, the authors manipulated neuron activity, using a viral vector to express an altered glycine receptor in the hippocampus on one side of the brain. When they injected mice intraperitoneally with a selective ligand that blocks this receptor, neuronal firing slackened on that side, and CSF flow ebbed. As a result, a dextran tracer injected into the CSF perfused more slowly on the silenced side of the brain (see image above). The same neuronal activity-CSF relationship held in mice during natural sleep.

If neuronal firing powers the entry of solutes to the brain, does it also affect their exit? To test this, the authors injected a dextran tracer into sleeping mice and allowed it to perfuse the brain for three hours before they silenced neurons. Four hours later, the silenced side of the brain had retained more tracer (see image at right).

Finally, the authors demonstrated that amping up neuronal firing could drive ion waves. For this experiment, they expressed an optogenetic ion channel in mouse hippocampus, turning it on by shining red light through the skull in a 1 Hz delta or 8 Hz theta rhythm. Theta rhythms occur during REM sleep. Regardless of which rhythm they used, as neurons fell into sync, more tracer entered the brain.

“These results … open up a new possibility to enhance brain clearance by harnessing neural dynamics,” the authors suggested.

Waving In Solutes. Labeled ovalbumin (aqua) entered the brain faster after 40-Hz stimulation (middle right) than after no (left), 8 Hz (middle left), or 80 Hz (right) stimulation. [Courtesy of Murdock et al., Nature.]

If slow sleep waves stimulate CSF flow, how about faster gamma waves? To investigate, first author Mitchell Murdock in Tsai’s group injected fluorescently labeled ovalbumin into the CSF of 6-month-old 5XFAD mice, then subjected the awake mice to a one-hour 40-Hz sound and light show. Four times as much ovalbumin reached their cortices as in mice exposed to 8 or 80 Hz stimulation, or no stimulation.

CSF outflow responded similarly. When the scientists burned a small hole in a blood vessel with a laser to allow labeled dextran to leak out into the gray matter, one hour of 40 Hz stimulation cleared up the mess 50 percent faster than in control mice. A third more amyloid reached cervical lymph nodes after 40-Hz stimulation than in control mice.

How could 40 Hz entrainment affect CSF flow? In part, perhaps, by making cerebral blood vessels dilate and contract more vigorously. The scientists measured vessels labeled with fluorescent dextran through a cranial window, finding that 40 Hz stimulation boosted the number of large arterial pulses by about half.

Neurons may do that via vasoactive intestinal peptide, the authors reported. After 40 Hz stimulation, interneurons released more VIP, which is known to regulate blood flow (Lee et al., 1984). With VIP interneurons inhibited, 40 Hz stimulation no longer affected arterial pulses or amyloid clearance, implicating VIP as a link between neurons and arteries. Some of this work was presented at the 2021 AD/PD conference (Apr 2021 conference news).

40 Hz rhythms normally mark the waking brain, though some studies have seen them during sleep, as well (Bergel et al., 2018; Laurino et al., 2019). It is unclear why both waking and sleeping firing rhythms seemed to promote CSF flow. Nedergaard and Hablitz speculated that different brain regions might have distinct “tuning frequencies” of stimulation that drive CSF perfusion and waste clearance. “Understanding the physiology and drivers of localized waste clearance in the brain could be the key that unlocks the therapeutic potential of the glymphatic system,” they suggested.—Madolyn Bowman Rogers.

Comments

1. • Ksenia Kastanenka
     Harvard Medical School, Massachusetts General Hospital
   • Posted: 21 Mar 2024

   Fantastic work by both groups. The work by Jonathan Kipnis and colleagues could provide an additional explanation for why we saw plaque clearance in APP/PS1 mice after optogenetic rescue of slow waves during sleep.

   References:

   Zhao Q, Maci M, Miller MR, Zhou H, Zhang F, Algamal M, Lee YF, Hou SS, Perle SJ, Le H, Russ AN, Lo EH, Gerashchenko D, Gomperts SN, Bacskai BJ, Kastanenka KV. Sleep restoration by optogenetic targeting of GABAergic neurons reprograms microglia and ameliorates pathological phenotypes in an Alzheimer's disease model. Mol Neurodegener. 2023 Dec 1;18(1):93. PubMed.

   View all comments by Ksenia Kastanenka

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References

News Citations

1. Flash! Beep! Gamma Waves Stimulate Microglia, Memory 15 Mar 2019
2. Gamma Waves Synchronized by Light: Good for Synapses, Memory? 17 May 2019
3. Brain Drain—“Glymphatic” Pathway Clears Aβ, Requires Water Channel 16 Aug 2012
4. Spinal Fluid Flush: Visualizing the Brain Drain With MRI 1 Mar 2013
5. Deep Sleep Makes Waves for CSF 1 Nov 2019
6. Does Synchronizing Brain Waves Bring Harmony? 14 Apr 2021

Series Citations

1. Opto- and Pharmacogenetics: Young Field Gives Researchers New Control 3 Nov 2012

Research Models Citations

1. 5xFAD (C57BL6)

Paper Citations

1. Hablitz LM, Vinitsky HS, Sun Q, Stæger FF, Sigurdsson B, Mortensen KN, Lilius TO, Nedergaard M. Increased glymphatic influx is correlated with high EEG delta power and low heart rate in mice under anesthesia. Sci Adv. 2019 Feb;5(2):eaav5447. Epub 2019 Feb 27 PubMed.
2. Hladky SB, Barrand MA. The glymphatic hypothesis: the theory and the evidence. Fluids Barriers CNS. 2022 Feb 3;19(1):9. PubMed.
3. Lee TJ, Saito A, Berezin I. Vasoactive intestinal polypeptide-like substance: the potential transmitter for cerebral vasodilation. Science. 1984 May 25;224(4651):898-901. PubMed.
4. Bergel A, Deffieux T, Demené C, Tanter M, Cohen I. Local hippocampal fast gamma rhythms precede brain-wide hyperemic patterns during spontaneous rodent REM sleep. Nat Commun. 2018 Dec 18;9(1):5364. PubMed.
5. Laurino M, Piarulli A, Menicucci D, Gemignani A. Local Gamma Activity During Non-REM Sleep in the Context of Sensory Evoked K-Complexes. Front Neurosci. 2019;13:1094. Epub 2019 Oct 15 PubMed.

Further Reading

News

1. Sleep and Brain Cleansing—Fresh Insights into Regulation and Disruption 19 May 2016
2. From ApoE to Zzz’s—Does Sleep Quality Affect Dementia Risk? 25 Oct 2013
3. Does Flashing Light Really Lower Cortical Amyloid? 10 Mar 2023