Research Pulse

Timekeeper: Glia?

January 24, 2023

Timekeeper: Glia?

Retinal circuitry & function in the day-and-night cycle

Background:

Seeing is among the most visceral and important experiences we humans make. At the molecular level, seeing is complex, not always reliable, and subject to a circadian rhythm regulating important metabolite oscillations such as melatonin and dopamine (Doyle S. et al., Vis Neuroscience, 2002).

But it might not stop there.

Evidence has been accumulating that makes neuroscientists re-evaluate how the retina is processing visual information at different times of the day.

Biological clocks

One of the most fascinating things about life is its incredible adaptability to environment and circumstance.

Among the most noticeable is life’s adaptation to the planetary movement of the earth, a rotation around its own axis which causes repetitive light-and-dark cycles on a 24h day. These diurnal or circadian rhythms are being felt and processed not only by mammals, but also by plants, fungi and even bacteria (Clodong S. et al., Mol Syst Biol., 2007).

That evolution would bring forth this circadian adaptation makes intuitive sense. Consider the fitness benefit of plant processes like leaf movement, growth, germination, oxygen exchange and photosynthetic ability, which need to be synchronized with the light cycles of its surrounding environment.

We humans are no exception, no matter if one is an early bird or a night owl, our bodies also work differently depending on the time of the day. Some examples of our circadian rhythm processes are:

  • melatonin levels (absent in daytime, regulate sleep)
  • body temperature (high in afternoon, low early morning)
  • cortisol plasma-levels (high in the morning, low at midnight)

This is of course old news. By investigating these well-known “circadian” patterns, researchers discovered a “master clock”, a biological structure in the brain called the suprachiasmatic nucleus (SCN) (Moore Y. et al., Brain Res, 1972). This tiny part of the hypothalamus is situated above the optic chiasm, the place where the optic nerves from both eyes cross. One part of the SCN is directly in connection with photosensitive retinal ganglion cells (RGC) and calibrated by direct photic input to induce gene expression changes, allowing the SCN to synchronize with day-night rhythms.

If you ever wondered how your body can handle trillions of cells working in concert to generate the beautiful you, synchronization is a big part of the answer.

Another, less understood part of the SCN is considered light-independent, running a roughly 24h rhythm even in total darkness (Kondratova et al., Nat Rev Neurosci., 2012).

The master pacemaker is located in the SCN of the hypothalamus and generates internal circadian rhythms in gene expression, electrophysiology and hormone secretion; direct projection from the retina transfers information about light/darkness to the SCN, which synchronizes a phase of SCN rhythms with the outside world. (Kondratova et al., Nat Rev Neurosci., 2012)
The master pacemaker is located in the SCN of the hypothalamus and generates internal circadian rhythms in gene expression, electrophysiology and hormone secretion; direct projection from the retina transfers information about light/darkness to the SCN, which synchronizes a phase of SCN rhythms with the outside world. (Kondratova et al., Nat Rev Neurosci., 2012)

Additionally, the SCN “master clock” exerts control over rhythmic processes all throughout the body, namely “peripheral clocks” or oscillators. Almost every tissue has rhythmic processes, including the adrenal gland, the lungs, pancreas, liver and the retina itself.

But how is biological rhythmicity even regulated?

Fluctuating clock genes

Regulation of gene expression, the process of when to produce which kind of proteins, can be understood in the abstract as a huge optimization problem for any organism. It’s a tough supply and demand system, optimized by evolution to reduce waste and increase efficiency, depending on cell type. Timing is essential.

To coordinate cellular processes throughout the day, transcription-translation feedback loops involving the heterodimerization of transcription factors BMAL1 and CLOCK (Takahashi JS, Nat Rev Genet., 2017) play a critical role, along with a host of downstream targets called clock-controlled genes (CCGs), as well as chromatin modifiers.

To go into detail would explode the length of this article, so we will just highlight some key points:

The mammalian circadian clock mechanism is cell autonomous and composed of a transcription-translation negative feedback loop. These clocks are distributed throughout the body and regulate tissue-specific rhythmic functions.

The core circadian transcriptional regulators drive gene expression rhythms in thousands of genes. CLOCK:BMAL1 target genes in the mouse liver regulate genes in all fundamental metabolic pathways suggesting that the clock system in intimately embedded in cellular metabolism.

Circadian activators and repressors recruit a wide array of chromatin modifiers leading to dynamic changes in the poise of the genome with time of day.

RNAPII is recruited and initiated genome-wide in a circadian manner in the mouse liver leading to genome-wide circadian changes in histone modifications.

Circadian CLOCK:BMAL1 gene targets are directly linked to metabolism, immune function, cell proliferation, cancer and signaling. —
Takahashi JS, Nat Rev Genet., 2017

The circadian gene network in mammals. At the core, CLOCK and BMAL1 activate the Per1, Per2, Cry1 and Cry2 genes, whose protein products interact and repress their own transcription, reorganize chromatin and impact translation. (Takahashi JS, Nat Rev Genet., 2017)
The circadian gene network in mammals. At the core, CLOCK and BMAL1 activate the Per1, Per2, Cry1 and Cry2 genes, whose protein products interact and repress their own transcription, reorganize chromatin and impact translation. (Takahashi JS, Nat Rev Genet., 2017)

Rhythmic expression of clock genes drives the transcription of thousands of clock-controlled genes (CCGs) that are not directly involved in modulating the circadian rhythm itself but generate rhythms in physiological processes in the brain and peripheral tissues, including the retina. — Riccitelli S. et al., Scientific reports, 2022

Oscillatory processes are very important in many tissues, but especially in retinal physiology and function. Circadian clock neurons in the retina generate daily rhythms via cell-autonomous autoregulatory clock gene networks (Ruan GX. et al., PNAS, 2006). Critical processes for vision, like photoreceptor renewal (Devera C. et al., Yale J Biol Med., 2019) and disc shedding (Chuang JZ et al., Cell, 2007) are coordinated via circadian processes, as well as melatonin and dopamine production (Doyle S. et al., Vis Neuroscience, 2002) which impact our awake/sleep cycle.

Given this dramatic physiological impact on the “hardware” of our retina, does that mean that our visual signal processing “software” is impacted by the time of the day too?

Glial influence on circadian firing rate

In a landmark paper, researchers from Harvard used a BMAL1 knockout mice to investigate this question. They discovered that their mice had altered electroretinogram b-wave recordings (a diagnostic marker) under light-adapted conditions.

Defective retinal electrical activity in response to light in Bmal1 knockout mice, but not SCN-lesioned wildtype mice (Storch KF. et al., Cell, 2007)
Defective retinal electrical activity in response to light in Bmal1 knockout mice, but not SCN-lesioned wildtype mice (Storch KF. et al., Cell, 2007)

Since then, multiple publications have found that perturbation of the circadian processes in retinal cells, both neurons and glia, can impact not only visual processing, but also behavior such as locomotor activity or cognitive function in mice (Tso CF. et al., Current Biology, 2017 & Barca-Mayo O. et al., Nature communication, 2017 & Brancaccio M. et al., Science, 2019).

Yet teasing apart which of the many different cell types within the retina contributes not only to circadian regulation, but actually shapes neuronal output of retinal ganglion cells, has been difficult because of RGC diversity.

There is a remarkable diversity of RGCs and the various subtypes have unique morphological features, distinct functions, and characteristic pathways linking the inner retina to the relevant brain areas. A number of psychophysical and electrophysiological tests have been refined to investigate this large and varied population of RGCs — Kim US. et al., Frontiers in Neurology, 2021

One way of classification for RGC is functional, based on their response to light at certain zeitgeber timepoints. For example, ON RGCs increase firing in response to light increments whereas OFF RGCs increase firing in response to light decrements.

Because of the large diversity, finding nuanced functional differences requires measuring thousands of RGC simultaneously, as a study has recently undertaken after the very subtle conditional deletion of the Bmal1 gene in GLAST-positive glial cells (Riccitelli S. et al., Scientific reports, 2022)

Functional characterization of over 20000 ON, ON–OFF and OFF type RGCs detected from responses to full-field flashes in Ctrl and Bmal1cKO retinae under mesopic conditions. ON RGCs (upper panels) increase their rate of discharge when the light illuminates in the centre. However, when the surround field is illuminated, the ON RGCs are suppressed. OFF RGCs respond when the light turns off (lower panels). ON/OFF cells are triggered briefly when the light turns on or off (Riccitelli S. et al., Scientific reports, 2022)
Functional characterization of over 20000 ON, ON–OFF and OFF type RGCs detected from responses to full-field flashes in Ctrl and Bmal1cKO retinae under mesopic conditions. ON RGCs (upper panels) increase their rate of discharge when the light illuminates in the centre. However, when the surround field is illuminated, the ON RGCs are suppressed. OFF RGCs respond when the light turns off (lower panels). ON/OFF cells are triggered briefly when the light turns on or off (Riccitelli S. et al., Scientific reports, 2022)

Using this experiment setup, the authors could show that glial Bmal1 influences the light response amplitude and firing rate of RGCs across different times of the day and has effects on luminance (contrast switching) dependent RGCs polarity preference (shown below).

Mean light luminance polarity shift for OFF preferential RGCs is altered upon glial BMAL1 deletion. (Riccitelli S. et al., Scientific reports, 2022)
Mean light luminance polarity shift for OFF preferential RGCs is altered upon glial BMAL1 deletion. (Riccitelli S. et al., Scientific reports, 2022)

These results are somewhat surprising, given that the conditional knockout of BMAL1 in a small subset of glial cells retains the ON, ON–OFF, and OFF subtypes and does not lead to noticeable morphological alterations or inflammatory processes that could explain an altered retinal function, which the authors attribute to Müller glia cells.

[…] our results indicate, for the first time, the involvement of glial cells (astrocytes and Müller cells) in the regulation of retinal function throughout the day. — Riccitelli S. et al., Scientific reports, 2022

While encouraging, the study also shows that to delineate all the contributing factors of this complex, time-dependent environment influencing neuronal processing in the retina (and with it certain behaviors and associated diseases) will likely take a long time.

Conclusion

Circadian rhythms are among the strongest known biological adaptations that living on a rotating planet brought with it.

The fact that many biological clocks tick inside our cells and shape the function of our body in harmony with the day-and-night cycle might be soothing to some or distressing to others, especially people who have to work at odd times of the day and feel the associated health impact.

However, the idea that not only our body, but also our senses and even our cognitive processes are impacted by our particular situation in the solar system has something eerie. Or maybe that is just me.

In any case, further studies are needed to delineate the impact of different cell types and circadian physiological processes on our retinal circuitry.

All what can be said at this point:

It seems clear that there is more influence on the retina’s neuronal output than meets the eye.

. . .

References:

Moore Y. et al., Brain Res, 1972

Doyle S. et al., Vis Neuroscience, 2002

Storch KF. et al., Cell, 2007

Clodong S. et al., Mol Syst Biol., 2007

Ruan GX. et al., PNAS, 2006

Kondratova et al., Nat Rev Neurosci., 2012

Takahashi JS, Nat Rev Genet., 2017

Tso CF. et al., Current Biology, 2017

Barca-Mayo O. et al., Nature communication, 2017

Brancaccio M. et al., Science, 2019

Devera C. et al., Yale J Biol Med., 2019

Kim US. et al., Frontiers in Neurology, 2021

Riccitelli S. et al., Scientific reports, 2022

Copyright:

Featured articles may include proprietary company information on products or research. For educational and other non-commercial purposes, you are allowed to share (copy and redistribute in any medium or format) & adapt (remix, transform, and build upon) the material as long as you give proper attribution (see also: CC BY-NC 3.0 license)

Declaration of interest:

The author is an employee at 3Brain.

The rich text element allows you to create and format headings, paragraphs, blockquotes, images, and video all in one place instead of having to add and format them individually. Just double-click and easily create content.

What’s a Rich Text element?

The rich text element allows you to create and format headings, paragraphs, blockquotes, images, and video all in one place instead of having to add and format them individually. Just double-click and easily create content.

Text Source

How to customize formatting for each rich text

Headings, paragraphs, blockquotes, figures, images, and figure captions can all be styled after a class is added to the rich text element using the "When inside of" nested selector system.

What’s a Rich Text element?

The rich text element allows you to create and format headings, paragraphs, blockquotes, images, and video all in one place instead of having to add and format them individually. Just double-click and easily create content.

Citation textd sfd
Static and dynamic content editing link

A rich text element can be used with static or dynamic content. For static content, just drop it into any page and begin editing. For dynamic content, add a rich text field to any collection and then connect a rich text element to that field in the settings panel. Voila!

How to customize formatting for each rich text

  • Test

Headings, paragraphs, blockquotes, figures, images, and figure captions can all be styled after a class is added to the rich text element using the "When inside of" nested selector system.

*You can unsubscribe anytime with the link provided in the notifcation emails