Title: Intrinsic circadian timekeeping properties of the thalamic lateral geniculate nucleus
Chrobok L.
1*
, Pradel K.
1
, Janik M.E.
2
, Sanetra A.M.
1
, Bubka M.
2
, Myung J.
3,4
, Rahim A.R.
3
, Klich J.D.
1
,
Jeczmien-Lazur J.S.
1
, Palus-Chramiec K.
1
, Lewandowski M.H.
1*
1
Department of Neurophysiology and Chronobiology, Institute of Zoology and Biomedical Research,
Jagiellonian University in Krakow, Gronostajowa Street 9, 30-387 Krakow, Poland
2
Department of Glycoconjugate Biochemistry, Institute of Zoology and Biomedical Research,
Jagiellonian University in Krakow, Gronostajowa Street 9, 30-387 Krakow, Poland
3
Graduate Institute of Mind, Brain, and Consciousness, Taipei Medical University, 172-1 Sec. 2
Keelung Road, Da’an District, Taipei 106, Taiwan
4
Brain and Consciousness Research Centre, Taipei Medical University-Shuang Ho Hospital, Ministry of
Health and Welfare, 291 Zhongzheng Road, Zhonghe District, New Taipei City 235, Taiwan
* corresponding authors
Author contributions: LC conceived the study. LC and MHL supervised the study. LC and JM designed
protocols and interpreted results of the study. LC, JM and KP provided financial support. JM and ARR
performed bioluminescence recordings and JM analysed and interpreted the data. LC, JDK and JSJ-L
collected brain sections for RT-qPCR and LC with AMS performed RNA isolation. MEJ and MB
designed, performed and analysed qPCR experiments. LC performed electrophysiological recordings
and analysed them with the use of custom-made tools provided by KP. LC and AMS carried out viral
injections and performed recordings combined with optogenetic stimulation, using the tools and
expertise provided by KP. LC, AMS, JSJ-L and KP-C performed the RNAscope experiment. LC wrote the
paper and all authors agreed to the final version.
Acknowledgements: We would like to thank the Department of Physiology and Toxicology of
Reproduction for the access to their laboratory equipment. Authors would also like to thank
Patrycjusz Nowik for the excellent animal care.
Funding: This work was financially supported by a project 'Sonatina 2' 2018/28/C/NZ4/00099 given
to LC and ‘Preludium 14’ 2017/27/N/NZ4/00785 to KP from the Polish National Science Centre. KP
was additionally supported by ‘Etiuda 8’ doctoral scholarship 2020/36/T/NZ4/00341. JM was
supported by the Taiwan Ministry of Science and Technology (109-2320-B-038-020, 109-2314-B-038-
071, 109-2314-B-038-106 -MY3, 108-2321-B-006-023-MY2, 107-2410-H-038-004-MY2), the Higher
Education Sprout Project by the Taiwan Ministry of Education (DP2-109-21121-01-N-01, DP2-110-
21121-01-N-01), and Taipei Medical University (TMU107-AE1-B15, 107-3805-003-110, 107TMU-SHH-
03).
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
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Conflict of interest: The authors declare no competing financial interests.
Ethics approval: Experiments were approved by the Local (Krakow) Ethical Commission and
performed in accordance with the European Community Council Directive of 24 November 1986
(86/0609/EEC) and the Polish Animal Welfare Act of 23 May 2012 (82/2012) and by the Institutional
Animal Care and Use Committee of Taipei Medical University (IACUC Approval No: LAC-2019-0118).
Data availability: The data that support the findings of this study are available from the
corresponding authors upon reasonable request.
ABSTRACT
Circadian rhythmicity in mammals is sustained by the central brain clock – the suprachiasmatic
nucleus of the hypothalamus (SCN), entrained to the ambient light-dark conditions through a dense
retinal input. However, recent discoveries of autonomous clock gene expression cast doubt on the
supremacy of the SCN and suggest circadian timekeeping mechanisms devolve to local brain clocks.
Here we use a combination of molecular, electrophysiological and optogenetic tools to evaluate
intrinsic clock properties of the main retinorecipient thalamic centre – the lateral geniculate nucleus
(LGN). We identify the dorsolateral geniculate nucleus (DLG) as a slave oscillator, which exhibits core
clock gene expression exclusively in vivo. Additionally, we provide compelling evidence for intrinsic
clock gene expression accompanied by circadian variation in neuronal activity in the intergeniculate
leaflet (IGL) and ventrolateral geniculate nucleus (VLG). Finally, our optogenetic experiments propose
the VLG as a light-entrainable oscillator, whose phase may be advanced by retinal input at the
beginning of the projected night. Altogether, this study for the first time demonstrates autonomous
timekeeping mechanisms shaping circadian physiology of the LGN.
Keywords: circadian clock, clock genes, lateral geniculate nucleus, light-entrainable oscillator, multi-
channel electrophysiology, PER2::LUC bioluminescence
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INTRODUCTION
Benefits and risks of the outside world undergo dramatic daily changes governed by the light-dark
cycle. Thus most, if not all, living organisms developed biological clocks, which enable them not only
to react to these cyclic events but also to predict them. In mammals, the central clock is localised in
the suprachiasmatic nuclei (SCN) of the hypothalamus (Hastings et al., 2018, 2019). Its timekeeping
properties are provided by clock cells maintaining a transcription-translation feedback loop (TTFL) of
core clock genes whose expressions are regulated by their own protein products in the period of
circa 24 h (Takahashi, 2017). Circadian rhythms on the molecular level are reflected in the daytime
rise in the electrical activity of the SCN neurons followed by their nocturnal silencing (Belle et al.,
2009; Colwell, 2011). Additionally, the SCN is best known for its photoentrainability, as it receives a
dense innervation from the retinal ganglion cells (heavily from these synthesising a ‘circadian
photopigment’ melanopsin) and responds to ambient light by both neurophysiological excitation and
phase shifts in clock gene expression (Beier et al., 2020).
The lateral geniculate nucleus (LGN) of the thalamus poses one of the main gateways for photic
information conveyed from the retina to the rest of the brain. Specifically, the LGN forms a complex
of three independent retinorecipient structures: (1) the dorsolateral geniculate nucleus (DLG), with
its thalamo-cortical neurons reaching the primary visual cortex and thus being directly involved in
image-forming vision (Sherman, 2005), (2) the intergeniculate leaflet (IGL) implicated in circadian
photoentrainment (Albrecht, 2012) (with one of the densest innervation by melanopsin cells; Brown
et al., 2010; Beier et al., 2020), but also non-photic behaviours such as modulation of mood, sleep,
spatial memory and food intake (Huang et al., 2019, 2021; Shi et al., 2019; Fernandez et al., 2020),
and (3) the ventrolateral geniculate nucleus (VLG) associated with visuomotor and other non-image
forming functions (Jeannerod & Putkonen, 1971; Legg & Cowey, 1977; Harrington, 1997). The VLG
can be further divided into a brainstem input processing medial part (VLGm), and a directly
retinorecipient lateral division (VLGl) (Niimi et al., 1963; Takatsuji & Tohyama, 1989; Kolmac &
Mitrofanis, 2000).
Early studies show daily rhythms in the multi-unit LGN activity dependent on the SCN (Inouye &
Kawamura, 1979) and an increased glucose utilisation in the LGN during the behaviourally active
night (Jay et al., 1985; Room & Tielemans, 1989). This is supported by the daily variability in its
spontaneous firing in vivo under urethane anaesthesia (Brown et al., 2011). Moreover, the IGL and
VLG have been reported to stay in the reciprocal connection with the central clock (Watts &
Swanson, 1987; Watts et al., 1987; Card & Moore, 1989; Moga & Moore, 1997; Moore et al., 2000).
Accumulating evidence questions the exclusive role of the SCN as the circadian clock and
demonstrates rhythmic clock gene expression in local brain clocks in the peripheral tissues
.CC-BY 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted May 7, 2021. ; https://doi.org/10.1101/2021.05.06.442920doi: bioRxiv preprint
(Granados-Fuentes et al., 2006; Guilding & Piggins, 2007; Herichová et al., 2007; Guilding et al., 2009,
2010; Zhang et al., 2014; Myung et al., 2019, 2018; Chrobok et al., 2020, 2021c; Northeast et al.,
2020; Chrobok et al., 2021d, 2021a; Paul et al., 2020). However, despite a striking day-to-night
difference in the main input to the LGN and a well-established involvement of the IGL in circadian
processes (i.e., the SCN clock resetting), little has been known about its circadian physiology
including intrinsic circadian timekeeping properties.
Here, we provide compelling evidence for intrinsic circadian timekeeping properties of the identified
structures of the LGN. We study their clock genes expression in vivo as well as in isolated slice culture
conditions. Additionally, we perform long-term multi-electrode array (MEA) recordings ex vivo
combined with timed optogenetic manipulation of the retinal input to elucidate circadian variability
in their neuronal firing and reveal the impact of photo-entrainment on their circadian activity
pattern. Our study identifies novel circadian extra-SCN oscillators, with the DLG displaying clock gene
expression exclusively in vivo, while the IGL and VLG possessing endogenous timekeeping
mechanisms (seen also ex vivo). These are additionally reflected in the nocturnal elevation of their
neuronal activity. Interestingly, we suggest that time of IGL peak firing is not shifted by retinal input
in contrast to the VLG, which we propose as a new light-entrainable oscillator.
MATERIALS AND METHODS
Ethical approval
Experiments on rats were approved by the Local Ethics Committee in Krakow and animals were
maintained and used according to Polish regulations and the European Communities Council
Directive (86/609/EEC). Procedures carried out on mice were reviewed and approved by the
Institutional Animal Care and Use Committee of Taipei Medical University (IACUC Approval No: LAC-
2019-0118). All possible efforts were made to minimise the number of animals used and their
sufferings.
Animals
This study was performed on 94 adult, male Sprague Dawley rats. Animals were kept under standard
12:12 h light-dark (LD) cycle, unless stated otherwise, with ad libitum access to food and water. Rats
were bred in the Animal Facility at the Institute of Zoology and Biomedical Research, Jagiellonian
University in Krakow, housed three to six per cage at 23 ± 2
o
C and 67 ± 3% relative humidity. Four
PERIOD2::LUCIFERASE (PER2::LUC) mice (RRID: IMSR_JAX:006852) were kept under a 12:12 light-dark
cycle at 21 ± 2°C and 59 ± 4% relative humidity in Taipei Medical University Laboratory Animal
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Centre. All procedures in darkness were performed in infra-red night vision goggles (Pulsar, Vilnius,
Lithuania).
Quantitative reverse transcription PCR
Tissue preparation
Three separate cohorts of animals were subjected to the RT-qPCR study: (1) 24 rats were culled in
four daily time points under LD cycle (n=6 each Zeitgeber time: ZT0, 6, 12 and 18), (2) 24 under DD at
four circadian time points (n=6 each circadian time: CT0, 6, 12 and 18), and (3) 30 under DD at six
circadian time points (n=5 for CT0, 4, 8, 12, 16 and 20). Two rats from the last group (one at CT8 and
one at CT16) were excluded from analysis due to outlying results in all parameters measured.
Animals culled in DD were moved to constant darkness conditions for two days before the cull. After
establishing the deep anaesthetic state with isoflurane (2 ml/kg body weight; Baxter, USA), rats were
decapitated and brains were quickly excised from the skull to ice-cold oxygenated (95% oxygen, 5%
CO
2
) preparation artificial cerebro-spinal fluid (ACSF), composed of (in mM): NaHCO
3
25, KCl 3,
Na
2
HPO
4
1.2, CaCl
2
2, MgCl
2
10, glucose 10, sucrose 125 and phenol red 0.01 mg/l. Brains were
trimmed, mounted in the chamber of a vibroslicer (Leica VT1000S, Germany) and cut in 250 μm thick
thalamic coronal sections. Bilateral LGNs were dissected using a scalpel and collected as whole, or
cut immediately above the IGL to separate the DLG from the IGL+VLG. Subsequently, the tissue was
flash frozen upon the dry ice and stored in −80
o
C, for up to a week. During the whole procedure, all
instruments and surfaces were treated with RNaseZAP (Sigma, Germany) to block ribonuclease
activity.
RNA isolation and RT-qPCR
Following the sampling, the RNA was extracted from the collected tissue with ReliaPrep RNA Tissue
Miniprep System (Promega, USA). Obtained RNA was stored at −80
o
C in RNase-free water, before
being processed to reverse-transcription using the High-Capacity RNA-to-cDNA Kit (Applied
Biosystems, USA). The normalised amount of RNA was used for each sample. Subsequently, qPCR
was carried out using PowerUp SYBR Green Master Mix (ThermoFisher Scientific, Lithuania) and
StepOnePlus Real-Time PCR System (Applied Biosystems). For transcript amplification, QuantiTect
primer assays (Qiagen, Germany) for selected genes were used. Per1, Arntl (Bmal1) and Nr1d1
(Reverbα) were chosen to measure clock genes and Gapdh served as a housekeeping gene. Results
were then analysed according to the Livak method (2
-ΔΔCT
) with Gapdh as reference gene and
presented as relative target gene expression (RQ), where RQ=1 indicates the ZT/CT0 mean.
PER2::LUC bioluminescence measurement
.CC-BY 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted May 7, 2021. ; https://doi.org/10.1101/2021.05.06.442920doi: bioRxiv preprint
