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Geniculohypothalamic GABAergic projections gate suprachiasmatic nucleus responses to retinal
input
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Lydia Hanna1, Lauren Walmsley1, Abigail Pienaar1, Michael Howarth1, Timothy M. Brown1*
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Faculty of Biology Medicine and Health, University of Manchester, UK
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*For correspondence:
AV Hill Building, University of Manchester, Oxford Road, Manchester, M13 9PT
[email protected]
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Running Title: Geniculohypothalamic signals gate SCN responses
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Keywords: circadian, electrophysiology, intergeniculate leaflet, mouse, channelrhodopsin
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TOC category: Neuroscience – behavioural/systems/cognitive
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Key Points Summary
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Visual input to the suprachiasmatic nucleus circadian clock is critical for animals to adapt
their physiology and behaviour in line with the solar day.
In addition to direct retinal projections, the clock receives input from the visual thalamus but
the role of this geniculohypothalamic pathway in circadian photoreception is poorly
understood.
Here we develop a novel brain slice preparation which preserves the geniculohypothalamic
pathway to show that GABAergic thalamic neurons inhibit retinally-driven activity in the
central clock in a circadian time-dependent manner.
We also show that in vivo manipulation of thalamic signalling adjusts specific features of the
hypothalamic light response, indicating that the geniculohypothalamic pathway is primarily
activated by crossed retinal inputs.
Our data provide a mechanism by which geniculohypothalamic signals can adjust the
magnitude of circadian and more acute hypothalamic light responses according to time-ofday and establish an important new model for future investigations of the circadian visual
system.
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Abstract
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Sensory input to the master mammalian circadian clock, the suprachiasmatic nucleus (SCN), is vital in
allowing animals to optimise physiology and behaviour alongside daily changes in the environment.
Retinal inputs encoding changes in external illumination provide the principle source of such
information. The SCN also receives input from other retinorecipient brain regions, primarily via the
geniculohypothalamic tract (GHT), but the contribution of these indirect projections to circadian
photoreception are currently poorly understood. To address this deficit, here we established an in
vitro mouse brain slice preparation which retains connectivity across the extended circadian system.
Using multi-electrode recordings, we first confirm that this preparation retains intact optic
projections to the SCN, thalamus and pretectum and a functional GHT. We next show that
optogenetic activation of GHT neurons selectively suppresses SCN responses to retinal input, that
this effect exhibits a pronounced day-night variation and involves a GABAergic mechanism. This
inhibitory action was not associated with overt circadian rhythmicity in GHT output, indicating
modulation at the SCN level. Finally, we use in vivo electrophysiological recordings alongside
pharmacological inactivation or optogenetic excitation to show that GHT signalling actively
modulates specific features of the SCN light response, indicating that GHT cells are primarily
activated by crossed retinal projections. Together, our data establish a new model for studying
network communication in the extended circadian system and provide novel insight into the roles of
GHT-signalling, revealing a mechanism by which thalamic activity can help gate retinal input to the
SCN according to time of day.
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Abbreviations
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ChR2, Channelrhodopsin-2; GAD2, glutamate decarboxylase 2; GHT, geniculohypothalamic tract; IGL,
intergeniculate leaflet; ipRGCs, intrinsically photosensitive retinal ganglion cells; OC, optic chiasm;
OT, optic tract; PCA, principle component analysis; PON, pretectal olivary nucleus; SCN,
suprachiasmatic nucleus; vLGN, ventral lateral geniculate nucleus; vSPZ, ventral subparaventricular
zone; ZT, Zeitgeber time.
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Introduction
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Visual input to the suprachiasmatic nucleus (SCN) circadian clock plays an essential role, allowing
mammals to optimise physiology and behaviour across the solar day. This function is supported by
direct retinal projections to the SCN from intrinsically photosensitive, melanopsin-expressing, retinal
ganglion cells (ipRGCs; Hattar et al., 2006). While ipRGCs are thus essential for circadian
photoentrainment (Güler et al., 2007; Chen et al., 2011), the SCN is also reciprocally connected to
several other ipRGC target regions: the thalamic intergeniculate leaflet/ventral lateral geniculate
nucleus (IGL/vLGN) and the pretectal olivary nucleus (PON) (Watts et al., 1987; Mikkelsen, 1992;
Kalsbeek et al., 1993; Morin et al., 1994; Horvath, 1998; Moore et al., 2000).
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So far, the IGL/vLGN projection to the SCN (the geniculohypothalamic tract; GHT) is the only
component of this extended circadian system that has been investigated in detail. IGL/vLGN cells are
primarily GABAergic and express a variety of peptide co-transmitters, with cells expressing
neuropeptide Y (NPY) providing a major (but not exclusive) contribution to the GHT (Moore & Card,
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1994; Morin & Blanchard, 1995; Morin & Blanchard, 2001). In line with the presence of substantial
input to the IGL/vLGN from the major brain arousal systems (Moore & Card, 1994), at present, the
best-defined role for the GHT is in mediating circadian responses to arousal-inducing (‘non-photic’)
stimuli (Harrington, 1997; Morin & Allen, 2006). Hence, circadian responses to non-photic stimuli
(which alter the timing of rodent behavioural rhythms in a manner essentially opposite to light) are
blocked by lesions of the GHT (Johnson et al., 1988; Janik & Mrosovsky, 1994; Wickland & Turek,
1994; Marchant et al., 1997) and can be mimicked by electrical stimulation of the GHT (Rusak et al.,
1989), in vivo/in vitro application of NPY (Shibata & Moore, 1993; Huhman & Albers, 1994; Huhman
et al., 1996) or GABA agonists (Smith et al., 1989; Gillespie et al., 1996; Huhman et al., 1997; Novak
et al., 2004).
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By contrast, despite the substantial retinal input to the IGL/vLGN (and its connections to the visual
centres in the pretectum), the contribution of the GHT to circadian photoreception remains
uncertain. Data from studies using pharmacological manipulation of either GABA (Gillespie et al.,
1996; Gillespie et al., 1997; Novak & Albers, 2004) or NPY signalling suggest that neurochemicals
released from the GHT could actively inhibit light-driven phase shifting (Yannielli & Harrington, 2000;
Lall & Biello, 2002, 2003; Yannielli et al., 2004). The extent to which these reported effects reflect
the physiological contribution of the GHT to photic phase-resetting or whether GABA and/or NPY
released from the GHT directly modulates light-evoked firing within the SCN remains uncertain,
however. This is especially true in the case of GABA signalling, where the pharmacological
approaches used above are unable to specifically distinguish effects arising due to GHT-derived
GABA release from those due to the action of local SCN neurons or other long-range inputs.
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Moreover, while lesion studies are equivocal as to how the GHT influences circadian photoreception,
on balance, these in fact seem to favour the view that circadian light responses are diminished,
rather than enhanced without a functional GHT (Harrington & Rusak, 1986; Pickard et al., 1987;
Johnson et al., 1989; Maywood et al., 1997; Edelstein & Amir, 1999a, b; Morin & Pace, 2002; Muscat
& Morin, 2006; Evans et al., 2012). Similarly, existing attempts to define the nature of the visual
signals conveyed by the GHT provide no clear consensus as to whether these are primarily activated,
suppressed or unresponsive to increases in illumination (Harrington & Rusak, 1989; Zhang & Rusak,
1989; Thankachan & Rusak, 2005; Blasiak & Lewandowski, 2013).
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Here then, we set out to resolve these questions as to how the GHT influences SCN visual
processing. We first generate and validate an in vitro model that retains an intact extended circadian
system, including a functional GHT. We next use this model, alongside optogenetic manipulations, to
define the impact of GHT signals on SCN responses to optic input. Finally, we perform in vivo
recordings coupled with local pharmacological inhibition or optogenetic excitation to reveal the
physiological impact of the GHT on light-evoked neural activity within the SCN.
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Materials and Methods
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Animals
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All animal use was in accordance with the Animals, Scientific Procedures, Act of 1986 (UK) and
received institutional ethics committee and UK Home Office approval. In vitro electrophysiological
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experiments were performed on adult (40-130 day old) male and female Opn4+/tau-lacZ mice and their
wild-type littermates (Hattar et al., 2006). For optogenetic control of GABAergic neurons, glutamate
decarboxylase 2 (GAD2)-IRES-Cre mice (Taniguchi et al., 2011) were crossed with a second line (Ai32)
exhibiting Cre recombinase-dependent Channelrhodopsin-2/EYFP (ChR2/EYFP) fusion protein
expression (Madisen et al., 2012). The resulting double heterozygous offspring thus exhibited
ChR2/EYFP expression restricted to GABAergic (GAD-2 expressing) neurons. In vivo
electrophysiological experiments were performed on adult male C57/Bl6 wild-type or GAD2-cre;
Ai32 mice under urethane anaesthesia. Animals were housed under a 12 h light:dark cycle at a
temperature of 22°C with food and water available ad libitum. Zeitgeber time (ZT) was used to
define the projected time of the SCN circadian clock, with lights-off defined as ZT 12.
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X-gal labelling and slice reconstruction
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In order to trace ipRGC projections to the extended circadian system, Opn4+/tau-lacZ mice were
euthanized with an overdose of urethane (intraperitoneal injection: 0.3ml of 20% urethane) and
were then transcardially perfused with 0.1 M phosphate-buffered saline (PBS; pH 7.4) followed by
cold, 4% paraformaldehyde (PFA) in PBS for 10 min. Brains were removed, post-fixed in 4% PFA
solution and then cryoprotected in 30% sucrose in PBS overnight. Coronal sections (60 µm thick)
were then obtained using a freezing sledge microtome (8000 Sledge; Bright Instruments, UK). To
visualise β–galactosidase expression, X-gal staining was then performed as described previously
(Sakhi et al., 2014). In brief, sections were washed twice for 10 min in Buffer B (0.1 M PBS at pH 7.4,
2 mM MgCl2, 0.01% Na-desoxycholate and 0.02% [octylphenoxyl] polyethoxyethanol [IGEPAL]) and
then incubated for 18 h in staining solution (Buffer B with 5 mM potassium ferricyanide, 5 mM
potassium ferrocyanide, and X-gal (1 mg/ml, Bioline Reagents Ltd., UK)) at 37ᵒC in darkness.
Following X-gal staining, sections were washed twice for 5 min in 0.1 M PBS and mounted onto glass
slides. Nuclear fast red counterstain (Sigma-Aldrich, UK) was applied to the sections for 5 min,
followed by blot-drying, dehydration steps (in 50, 70 and 100% ethanol: 5 min each), and finally a
clearing agent was added for 5 min (HistoChoice, Sigma-Aldrich, UK). Slides were then mounted with
DPX (Sigma-Aldrich, UK) and coverslipped. Images of serial sections were acquired with a PlanApochromat (20x/0.80) objective using a Pannoramic 250 Flash II slide scanner (3DHISTECH,
Hungary). Resulting images of each brain section were then manually aligned and imported into
MATLAB (The Mathworks, Inc., USA) where RGB colour ratios were used to distinguish X-gal staining
from background (fast red) and produce a 3D map of relative ipRGC fibre/terminal density.
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Immunofluorescence processing
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For visualisation of ChR2 expression, GAD2-Cre; Ai32 mice were euthanized and brains were
cryoprotected and sectioned as indicated above for X-gal labelling studies (except in this case 50 µm
thick). Resulting brain sections were washed three times in 1% triton-X 100 in PBS (PBS-T) for 5 min
each and then incubated in blocking solution (10% normal donkey serum, 0.2% PBS-T) for 1 h at
room temperature (21ᵒC). Sections were subsequently incubated in chicken anti-GFP primary
antibody (Abcam, ab13970; 1:1000, diluted in blocking solution) overnight at 4˚C. After the
incubation period, sections were washed three times in 0.2% PBS-T for 15 min each and then
incubated in Alexa Fluor 488-AffiniPure donkey anti-chicken secondary antibody (Stratech, 703-545155-JIR; 1:5000, diluted in blocking solution) for 1 h at room temperature. Sections were washed
three times in PBS for 15 min, mounted onto glass slides with DAPI-containing Vectashield HardSet
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(Vector Labs, H-1500) and coverslipped. Epifluorescence images of immunostaining were then
acquired by Pannoramic 250 Flash II slide scanner as described above.
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In vitro multi-electrode recordings
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Mice were sacrificed by cervical dislocation followed by decapitation (ZT2-4). Brains were then
rapidly removed and placed into a custom-made adult mouse brain matrix (Zivic Instruments, USA).
The base of the brains (just rostral to the cerebellum) were cut at 30° off the coronal plane and
immediately mounted onto a metal stage and sectioned using a 7000 smz-2 vibrating microtome
(Campden Instrument, UK) in ice-cold (4°C) sucrose-based slicing solution composed of (in mM):
sucrose (189); D-glucose (10); NaHCO3 (26); KCl (3); MgSO4 (5); CaCl2 (0.1); NaH2PO4 (1.25);
oxygenated with 95% O2 / 5% CO2 mixture. Once the optic chiasm/rostral pole of the SCN became
visible we then cut a 600 μm thick section which contained the SCN, IGL/vLGN and PON. Only one
slice was produced per animal. The resulting slice was then transferred into a petri dish containing
oxygenated artificial cerebrospinal fluid (aCSF) composed of (in mM): NaCl (124); KCl (3); NaHCO 3
(24); NaH2PO4 (1.25); MgSO4 (1); glucose (10); CaCl2 (2); slices were then left to rest for 1 h at room
temperature (22°C).
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Multi-electrode array recordings from acute brain slices were performed as previously described
(Walmsley et al., 2015). Slices were placed, recording side down, onto 60pMEA100/30iR-Ti-gr
perforated multi-electrode arrays (pMEAs; Multi Channel Systems, MCS GmbH, Germany),
comprising 59 electrodes (site area: 707 µm2) spaced 100 µm apart with one dedicated low
impedance reference electrode. Perforated arrays were chosen due to their improved slice perfusion
rate, low signal-to-noise ratio and long-term recording stability (Reinhard et al., 2014). Confirmation
of optimal slice placement over the pMEA electrode sites was confirmed by overlaying images taken
with a GXCAM-1.3 camera (GX Optical, UK). Where relevant, anatomical placement was also
confirmed post hoc with X-gal staining (for Opn4+/tau-lacZ) or endogenous EYFP fluorescence (for
GAD2-Cre; Ai32) as discussed below.
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During recordings, slices were held in place by both the bottom flow suction (via the MEA
perforations) driven by a constant vacuum pump (MCS GmbH, Germany) and a weighted grid. The
pMEA recording chamber was continuously perfused with pre-warmed oxygenated aCSF (34 ± 1°C)
to both slice surfaces at a combined rate of 2.5–3 ml/min. For long-term recordings (>26 h), 0.001%
gentamicin (Sigma-Aldrich, UK) was added directly to the aCSF. Neural signals were acquired with
MC_Rack software as time-stamped action potential waveforms using a USB-ME64 system and
MEA1060UP-BC amplifier (MCS GmbH, Germany). Signals were sampled at 50 kHz (12.5 kHz for longterm recordings), high-pass filtered at 200 Hz (second order Butterworth) and multiunit spikes
crossing a threshold (usually set at -16.5 μV) were extracted for further analysis. In some cases we
also isolated single unit activity from these multiunit recordings, using principle components analysis
(PCA)-based spike sorting (Offline sorter V3; Plexon, USA). Reliable single unit isolation was
confirmed by reference to MANOVA F statistics, J3 and Davies-Bouldin validity metrics (Offline
sorter) and the presence of a distinct refractory period (>20ms for SCN neurons) in the interspike
interval distribution.
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For post-hoc histological analysis, slices were submerged in 4% PFA at room temperature for 40 min
and then kept overnight at 4ᵒC in 0.1 M PBS. In the case of X-gal staining, sections were then
additionally washed twice for 15 min in Buffer B and incubated overnight in staining solution at 37ᵒC
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in darkness (see solution details in X-gal labelling section above). All sections were subsequently
washed twice for 5 min in 0.1 M PBS and mounted onto glass slides with Fluoromount (SigmaAldrich, UK). Images of X-gal staining were acquired with ZEN digital imaging software (Carl Zeiss,
Germany) and EYFP fluorescence was captured on an Olympus BX51 upright microscope (4x/0.30
Plan Fln) using a Coolsnap ES camera (Photometrics) with MetaVue Software (Molecular Devices).
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Electrical and optogenetic stimulation
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To assess responses to optic input, retinal projections were activated by electrical stimulation of the
optic chiasm (OC) using two methods: either biphasic stimulation via a neighbouring pair of pMEA
electrodes visually confirmed to be situated within the OC (2500 mV, 100 µs duration, 0.5 Hz) or via
an independently positioned stimulating concentric bipolar electrode (FHC, USA; 0.3 - 1.5 mA, 200 µs
duration, 0.5 Hz). Similar stimulation parameters were used to activate the GHT (in this case with the
stimulating electrode positioned within the IGL-region at least ~200 μm lateral to the optic tract).
Stimuli were applied as single pulses (0.5 Hz) or as a train of five stimuli, separated at 20 or 40 Hz.
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For optogenetic stimulation, blue light flashes (5 - 10 ms duration, 0.5 Hz) were delivered by a 200
µm core fibre (465 nm LED, ~800 mW/mm2 at fibre tip; PlexBright; Plexon) placed 100-200 µm above
the slice surface over the IGL or SCN region. Based on the numerical aperture of the fibre (0.66), this
stimulus illuminates a ~500 μm diameter portion of the slice (although due to light scatter the
effective stimulated region will be larger). In order to assess functional convergence of retinal and
thalamic inputs in the SCN and GHT signalling mechanisms, optical stimulation of the IGL/vLGN was
synchronously delivered, or preceded pMEA electrical stimulation (2500 mV, 100 µs duration) of the
OC at various latencies (25 ms - 1 s, 10 – 60 s between each OC/GHT pair: 5 - 6 different latencies
were tested per experiment and delivered in an interleaved fashion). For long-term recordings of
thalamic evoked and spontaneous activity, we alternately stimulated optic input via a concentric
electrode placed in the OC (0.6 - 1.2 mA, 200 µs duration, 0.02 Hz) and directly activated GHT
GABAergic neurons via the optical fibre as above (5 ms duration, 0.02 Hz).
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Pharmacological manipulation
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Pharmacological separation of the synaptic components of evoked responses was achieved by bath
application of ionotropic NMDA (D-(-)-2-Amino-5-phosphonopentanoic acid; D-AP5, 50 µM) and
AMPA/kainate (6-Cyano-7-nitroquinoxaline-2,3-dione disodium; CNQX, 20 µM) glutamate receptor
blockers. Contributions of GABAergic transmission were evaluated by bath perfusion with (+)bicuculline (BIC, 20 µM), an antagonist at ionotropic GABAA receptors, and CGP55845 hydrochloride
(CGP, 3 µM), an antagonist at metabotropic GABAB receptors. At the end of all experiments, slices
were treated with bath application of N-Methyl-D-aspartic acid (NMDA, 20 μM) to confirm
maintained cell responsiveness, followed by tetrodotoxin citrate (TTX, 1 μM), to confirm that
acquired signals exclusively reflected Na+-dependent action potentials. All drugs were purchased
from Sigma-Aldrich (UK) or Tocris (UK), dissolved in ddH2O and kept as stock solutions at -20°C (with
the exception of BIC and CGP which were dissolved in DMSO) and were diluted to their respective
final concentrations in pre-warmed, oxygenated aCSF.
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Visual stimuli
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Visual stimuli were delivered and quantified as described previously (Walmsley & Brown, 2015). Full
field visual stimuli were generated via two LEDs (λmax 405 nm; half-width: ±7 nm; Thorlabs, USA),
controlled via LabVIEW (National Instruments, USA) and neutral density filter wheels (Thorlabs,
USA). Light was supplied via flexible fibre optic light guides (Edmund Optics, UK; 7 mm diameter)
terminating in internally reflective plastic cones that fit snugly over the mouse’s eye to prevent offtarget effects to stimulation of the opposing eye. Light measurements (at the level of the mouse
eye) were performed using a calibrated spectroradiometer (Bentham instruments, UK), with
effective photoreceptor excitations calculated using established measures (Govardovskii et al., 2000;
Jacobs & Williams, 2007; Walmsley et al., 2015). Owing to our choice of wavelength, apparent
brightness of our stimuli differed by no more than 0.2 log units for any of the known mouse opsins,
with values reported in this manuscript reflecting effective irradiance for rod opsin.
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In vivo multi-electrode recordings
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Mice were removed from their housing environment 1-2h before lights off (ZT10-11), anaesthetised
by intraperitoneal injection of urethane (1.55g/kg in 0.9% physiological saline) and then placed in a
stereotaxic frame (SR-15M; Narishige International Ltd., UK), where their temperature was
maintained at 37˚C using a homeothermic mat (Harvard Apparatus, UK). The surface of the skull was
exposed and two holes were drilled using stereotaxic co-ordinates (0.95 mm lateral to, and 0.3 mm
posterior to bregma for SCN recording electrode; 2.3 mm lateral to, and 3.5 mm posterior to bregma
for IGL probe) obtained from the mouse atlas (Paxinos & Franklin, 2001). Pupils were dilated with
topical application of atropine (1% in saline) and a thin coating of mineral oil was then applied to
prevent drying of the cornea (both sourced from Sigma-Aldrich, UK).
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For SCN recordings 32-channel electrodes were then coated with fluorescent dye (DiI; Invitrogen,
UK) and inserted into the brain 0.95 mm lateral, and 0.3 mm caudal to bregma (angled at 9° relative
to the dorsal-ventral axis). We employed two different electrode configurations for these studies:
Buszaki 32L (Neuronexus, USA) or 2x16Parallel (Cambridge Neurotech; UK) both of which produced
equivalent data. Buszaki32L consisted of four shanks (200 μm spacing) each with eight closely
spaced recordings sites in diamond formation (inter-site distance of 22 - 42 μm; site area: 160 μm2).
The 2x16Parallel electrodes had two shanks (250 μm spacing) each with two rows of 8 recording
sites (25 μm spacing; site area: 160 μm2). For manipulation of thalamic activity a second neural
probe (see details below), again coated in DiI, was then inserted into the brain 2.3 mm lateral, and
3.5 mm caudal to bregma (angled at 16° relative to the dorsal-ventral axis) such that the tip of the
electrode reached the IGL-region.
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In all cases, electrodes were then lowered into position using a fluid-filled micromanipulator (MO-10,
Narishige International Ltd., UK). After allowing at least 30 min for neural activity to stabilise
following probe insertion, wideband neural signals were acquired using a Recorder64 system
(Plexon), amplified (x3000) and digitized at 40 kHz. Timestamped multiunit spikes (1 ms segments of
data) were then extracted from these recordings by threshold crossing (-40 µV) for subsequent
analysis.
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At the end of these in vivo experiments, brains were removed and post-fixed in 4% PFA and then
cryoprotected and sectioned (100 µm) as described above for X-gal labelling studies. DiI-marked
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probe position (and where relevant endogenous GAD-driven EYFP fluorescence) was then visualised
and captured on an Olympus BX51 upright microscope (4x/0.30 Plan Fln) using a Coolsnap ES camera
(Photometrics) with MetaVue Software (Molecular Devices).
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Pharmacological inactivation of the visual thalamus
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For these experiments we implanted a 16-site linear recording array (100 μm spacing; site area: 177
μm2; E16-20mm-100-177; Neuronexus, USA) with a drug cannula (outer diameter 165μm; fused to
the underside of the array) protruding 100 μm ventral to the probe tip into the IGL/vLGN region. To
facilitate drug infusion into the visual thalamus, the cannula underlying the recording probe (100 µm
aperture) was connected via flexible narrow bore tubing to a syringe pump (Harvard Apparatus,
Cambridge, UK) preloaded with muscimol (1 mM, Sigma).
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After allowing neural activity to stabilise, we then evaluated baseline visual responsiveness across
the SCN and visual thalamus. Mice were maintained in darkness and 5 s light steps were applied in
an interleaved fashion to contralateral and/or ipsilateral eyes for a total of 10 repeats at
logarithmically increasing intensities spanning 11.4 - 15.4 log photons/cm2/s (inter-stimulus interval:
20 s).
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Once we had collected this baseline data we then began to infuse muscimol into the thalamus in 1 μl
increments (delivered over 1 min) while monitoring responses to visual stimuli (5 s binocular light
steps 15.4 log photons/cm2/s). Drug onset (observed after nominal injection volumes of 2-3 μl) was
easily identifiable by a rapid loss of spontaneous and evoked neural activity, alongside a small
increase in electrical noise. Following confirmation of abolition of thalamic activity by muscimol
application, mice were then dark adapted (30 min) and the protocol above was repeated. In no case
did we observe a reversal of drug effects during our recording sessions.
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Optogenetic activation of the visual thalamus
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These experiments were performed in GAD2-cre; Ai32 mice. We implanted a 32-site recording array
(3 parallel rows of 10-12 sites at 50 μm spacing; site area: 177 μm2; OALPPoly3; Neuronexus) into
the thalamus with an optical fibre (110 μm core; 0.66 Numerical Aperture) terminating 200μm above
the dorsal-most recording site. For optogenetic stimulation, the optical fibre was connected to a
465nm PlexBright LED (as above), providing ~630mW/mm2 light energy at the fibre tip.
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After allowing neural activity to stabilise, we first confirmed that we were able to manipulate
GABAergic neuronal activity within the visual thalamus by monitoring multiunit responses to a train
of 10ms light flashes (100 repeats at 0.5Hz). We then evaluated neural responses across the SCN and
visual thalamus to bright light steps (15.4 log photons/cm2/s) targeting either the contralateral,
ipsilateral or both eyes in the presence or absence of repeated optogenetic stimulation (5s trains of
10ms flashes at 5, 10 and 20Hz). Each test stimulus was repeated 10 times in an interleaved fashion
(with 40s inter-stimulus interval).
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Data analysis
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Interpretation of multiunit data
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Here we evaluate neural responses to a range of stimulus conditions by monitoring ensemble
activity detected across multiple recording electrodes within a single preparation. Multiunit
recording has been used extensively to monitor SCN activity (Kubota et al., 1981; Tcheng & Gillette,
1996; Gribkoff et al., 1998; Yamazaki et al., 1998; Mrugala et al., 2000; Tousson & Meissl, 2004;
Brown et al., 2006; VanderLeest et al., 2007; Nakamura et al., 2008) and provides a reliable
indication of overall population activity within the nucleus (Schaap et al., 2003; Brown & Piggins,
2009) that matches well that attained using other single-cell recording approaches (Green & Gillette,
1982; Groos & Hendriks, 1982; Shibata et al., 1982; Schaap et al., 1999; Pennartz et al., 2002; Itri et
al., 2005; Nygård et al., 2005). Nonetheless, an important first consideration in interpreting the data
from our multi-electrode recordings is the size of the neural ensemble such recordings represent
and the extent to which signals detected at different electrodes can be considered independent.
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Based on the typical peak SCN multiunit firing rates we detect in vitro for recordings spanning a full
circadian cycle (mean ± SD: 4.4±6.4 spikes/s across each channels most active 1hr epoch; n=144
channels from 5 slices), relative to equivalent data obtained for single SCN cells (mean = 2.5 spikes/s;
(Brown & Piggins, 2009), we consider it highly unlikely that any single electrode samples from more
than 7 neurons. Given the small size and dense packing of SCN cells (diameter <10μm; average intersoma distance ~14 μm; Abrahamson & Moore, 2001), this upper limit of 7 neurons would be
contained within a hemisphere with a radius of 17 μm relative to the centre of each recording site.
We thus consider it most unlikely that we detect cells further than 17μm from each electrode
(substantially smaller than the inter-electrode distance of these arrays: 100μm).
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Similarly, comparing the typical light evoked multiunit activity we detect in vivo (mean ± SD:
14.1±15.5 spikes/s for a 5s, 405nm light step providing 15.4 log photons/cm 2/s to both eyes; n=295
channels from 20 mice) relative to typical single cell responses to an equivalent stimulus (mean=9.7
spikes/s; Walmsley & Brown, 2015), we estimate our multiunit recordings derive from no more than
5 cells/channel. This suggests an upper limit to the listening range of each electrode of ~15 μm
(approximately half the inter-electrode distance for these arrays: 22-42μm).
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To provide additional confidence that the multiunit signals we record at neighbouring electrodes are
truly independent, we also calculated the incidence of synchronous spike detection (defined here as
spikes occurring within ±200μs) at adjacent channels across subsets of our in vivo or in vitro SCN
recordings. Since some SCN neurons are known to communicate using gap junctions (Long et al.,
2005), the results of this analysis should be considered as an upper bound to the extent to which we
may detect the same neuron at more than one electrode.
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In the case of in vitro recordings, we analysed data from 794 pairs of horizontally or vertically
adjacent channels that showed detectable neural activity (from 5 slices recorded over a full circadian
cycle) and found that the incidence of synchronous spikes was very low (median = 1.2% of spikes at
each channel, interquartile range=0.4-2.9% of spikes). An equivalent analysis performed on data
from in vivo recordings (560 pairs of adjacent channels from 10 recordings) revealed a broadly
similar incidence of synchronous spikes (median = 2.1% of spikes at each channel, interquartile
range=1.2-3.7% of spikes). Thus for both recording modalities, we consider the signals detected at
individual channels as almost exclusively representing the activity of distinct groups of neurons.
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Anatomical locations of recorded ensembles
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As indicated in the relevant sections above, placement of recording electrodes in all experiments
was confirmed by overlaid reconstructions of the known recording array geometry and histological
images (using ventricles and fibre tracts as landmarks and, where available, X-gal staining or EYFP
fluorescence). The anatomical extent of our recording arrays ensured that in many experiments
some recording sites were located outside our regions of interest. For most of the analysis presented
here, where electrode sites situated formally just outside these regions of interest exhibited
functional responses to stimulation of retinal afferents pathways (see below), such data was
included in our overall analysis. In particular, for hypothalamic recordings, retinally-driven responses
occasionally identified at recording sites located in the ventral subparaventricular zone (vSPZ), just
dorsal to the SCN, were also included in our analysis. Exceptions to the rule above were our analysis
of circadian rhythms in SCN, vSPZ and IGL/vLGN output (analysis presented in Fig. 3 and Fig. 6),
where we excluded data from sites located outside the borders of these regions evident in the
anatomical images. Small errors in our anatomical classification of recording site location here are
most unlikely to have influenced our conclusions. Hence, including or excluding data from sites
located at the projected anatomical borders of the regions in question produced essentially identical
results to those reported in this manuscript.
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Identification of responding channels
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Acute changes in neural activity evoked by electrical or optogenetic stimulations were classified as
excitatory or inhibitory when the average spike counts across multiple stimulus repeats (typically
substantially >100) respectively exceeded the upper or lower bounds of the 99% confidence limits
for prestimulus spike counts (within at least one 10ms bin <100ms after stimulus onset). For the
majority of data presented in this manuscript (fast excitatory responses which are relatively time
locked to the stimulus), representative multiunit data illustrating such responses are presented as
perievent histograms reporting spike probability per unit time relative to stimulus onset. The
exceptions here are data for GHT-driven inhibitory responses where perievent histograms are
instead converted to average firing rates per unit time across trials.
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Where relevant (analysis presented in Figs. 6 and 7), channels identified as responding to electrical
stimulation of the OC were further categorised as receiving modulatory input from the GHT. For this
analysis, channels were considered ‘GHT responsive’ when the OC-evoked response following prior
optogenetic stimulation of the GHT (at any latency) was significantly different (P<0.05) to the
response evoked following synchronous stimulation (paired t-tests with Bonferroni correction, based
on responses to at least 120 trials at each latency).
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For in vivo recordings, recorded ensembles at each site were considered light responsive when mean
firing rates over the course of 5s binocular light exposure (15.4 log photons/cm 2/s) were significantly
different to the firing rate in the 5s epoch immediately prior to light exposure (paired t-test from
responses to 10 trials).
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For analysis of responsiveness to pharmacological agents (NMDA; Fig. 3), channels were considered
responsive based on previously established criteria (Brown et al., 2008): when the mean firing rate
changed by more than 20% during the 3-8min after the start of drug application relative to pre-drug
levels. To account for the possible confounding effects of baseline drift, pre-drug firing was
extrapolated based on a linear fit to the mean firing rates observed over the 5 min epoch
immediately prior to the start of drug application.
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Analysis of multiunit responses
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Where we report average time-course data for excitatory responses in the presence and absence of
pharmacological blocking agents (Fig. 2.), we combined data from all channels identified (as above)
as responding across each of the 2-6 slices tested. For this analysis we first calculated, for each
channel, perievent response histograms (5ms bin size) for the 6min epochs immediately prior to
drug application and 3-9min immediately following the start of drug application (corresponding to
180 trials in either epoch). These histograms were then normalised by subtracting pre-stimulus spike
counts (averaged across the 50ms preceding stimulation) and divided by the maximal response
observed under baseline conditions. For comparison of pre- and post-drug responses the resulting
datasets were then analysed by two-way repeated measures (RM) ANOVA with Sidak’s test for
multiple comparisons (GraphPad Prism, GraphPad Software, USA).
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For quantification of the time-course and magnitude of SCN responses to GHT-stimulation we
produced peri-event histograms (responses to at least 300 trials, bin size=1ms) and smoothed with a
10ms boxcar filter. Latency was measured as time to the largest positive poststimulus deflection and
response magnitude measured as the percent change in spike probability relative the average spike
probability during the 50ms epoch preceding stimulation. The effects of pharmacological agents on
this latter parameter were compared by paired t-tests of equivalent analysis performed on
responses occurring 0-10 min prior to drug application vs. 5-15 min after the start of drug perfusion.
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For comparisons of activity evoked during combined GHT/OC stimulation (Figs 6 and 7), responses
were quantified as the change in spike probability between the 25ms epochs immediately before
and after OC stimulation. For analysis, the corresponding data for each channel was then normalised
according to the maximal response observed at any GHT-OC stimulation delay. For experiments
involving pharmacological blocking agents this normalisation was applied separately for baseline and
each of the drug treatments (25min epochs starting immediately prior to or 5 to 30 min after the
start of each 30min drug application; corresponding to 50 trials/epoch). Statistical significance was
assessed using two-way RM ANOVA and Sidak’s test, as above.
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For most analyses of in vivo visual response magnitude, the change in firing at each light responsive
channel (relative to the 5s epoch immediately prior to light stimulation) was normalised according to
the largest value obtained for that site under all stimulus conditions tested (i.e. across each
intensity/eye/degree of GHT manipulation). Changes in amplitude/sensitivity (Fig. 8), were then
assessed by fitting irradiance response relationships to the normalised data using 4-parameter
sigmoidal functions, with F-test (GraphPad) used to determine whether the fitted parameters
differed significantly between pre and post-muscimol application for each stimulus condition. Where
this analysis indicated significant differences in the irradiance response relationship pre- vs. postmuscimol for a given condition, we renormalised the two curves to have the same maximal value
and repeated the F-test (to specifically assess whether there were differences in sensitivity). For
assessment of changes in visual response amplitude as a function of GHT stimulation frequency (Fig.
9F), data were normalised separately for each eye and analysed by two-way RM ANOVA. Equivalent
data for LGN recordings are analysed and presented in raw (not normalised) form.
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Analysis of circadian rhythmicity
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For analysis of circadian rhythmicity in long-term hypothalamic recordings (data presented in Fig. 3),
data from each channel was considered to exhibit circadian variation when it could be better fit by a
sinusoidal function (constrained to have a period between 20 and 28h) vs. a first order polynomial
(calculated using custom MATLAB routines). Data from a small number of channels where we did not
detect multiunit activity (n=37/295 from 5 slices; primarily channels located in the optic tract or
over the 3rd ventricle) were excluded from this analysis. Of the remaining channels, identified best fit
functions described the data well (median±SD of Pearson’s r values = 0.81±0.16, n=258). For those
channels considered to exhibit circadian variation (median±SD of Pearson’s r values for sinusoidal
fit= 0.83±0.15, n=168) we next calculated the timing of peak firing. For the analysis presented in the
manuscript, multiunit time series were binned (60 s) and smoothed (2 h boxcar filter), with the ZT of
bin exhibiting maximal values taken as the timing of the peak.
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The phase and co-ordination of the daily peak in electrical activity was then calculated by Rayleigh
analysis using custom MATLAB scripts as described previously (Walmsley et al., 2015). Comparable
analysis performed using peak ZT’s estimated directly from the sinusoidal fits produced essentially
identical results. Differences in clustering strength and phasing were assessed by first linearising the
data such that, within each dataset, each observation was no more than 12h displaced from the
overall circular median (i.e. subtracting 24h from those observations that occurred >12h later and
adding 24h to those observations occurring >12h earlier than the median). We then applied
conventional analysis to identify differences in variance (Browne-Forsythe’s test) and median (MannWitney U-test).
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For other analysis of circadian variation presented here (Fig. 6), spontaneous or evoked responses at
each responding recording site within the region of interest were 1 h-binned and then normalised
according to the peak value observed across all bins. We then tested whether the resulting
normalised population datasets were better fit by a sinusoidal function (with 24h periodicity) or a
first order polynomial (via F-test; GraphPad). Where data were better fit by a sinusoidal function we
rebinned/normalised the data into 6h blocks and performed one-way RM ANOVA with Tukey’s post
hoc tests.
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Results
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Generation and validation of a functional in vitro model of the extended circadian system
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In order to better understand the physiological roles of GHT signals in modulating SCN responses to
retinal input, we first set out to develop an in vitro model which preserved functional connectivity
across the extended circadian system. Since projections between the SCN, IGL/vLGN and PON all run
via the optic tract (OT; Watts et al., 1987; Mikkelsen, 1992; Kalsbeek et al., 1993; Morin et al., 1994;
Horvath, 1998; Moore et al., 2000), we initially aimed to determine what angle and thickness of
brain section would be required to preserve OT-projections to all of these nuclei largely intact. To
achieve this aim, we exploited the fact that M1-type ipRGC projections specifically identify nuclei of
the extended circadian system in the mouse brain (Hattar et al., 2006). Using a selective reporter
mouse line, Opn4tau-LacZ, we were thus able to readily visualise retinal projections to all three nuclei in
X-gal stained coronal serial sections (Fig. 1A). After aligning the resulting sections, we then
generated a 3-dimensional map of M1-ipRGC central projections. Based on a flattened sagittal plane
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view of this projection map (Fig. 1B), we determined that a 600 µm thick brain section cut at 30° off
the coronal plane would preserve OT projections and large portions of the SCN, IGL/vLGN and PON
intact (for coronal plane reconstruction of the resulting slice see Fig. 1C).
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Using the results of this modelling we next prepared live brain sections from Opn4+/tau-LacZ mice, with
the aid of a custom matrix that allowed us to accurately block the brain at the required angle for
subsequent sectioning. From initial light microscopic visualisation of the resulting slices, we could
reliably (>90% of slices) observe a continuous OT extending far beyond the thalamus and easily
identifiable SCN, IGL/vLGN and pretectal regions (Fig. 2A), subsequently confirmed by post hoc X-gal
staining (Fig. 2B). We next set out to confirm that optic tract connectivity across these visual nuclei
remained functional in our slice preparation via pMEA recording.
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As expected, electrical stimulation of the optic chiasm (OC) in these angled slices reliably evoked
spike firing at subsets of recording sites located in the pretectum (Fig. 2C,D; n=21 responding sites
identified in 3 slices), visual thalamus (Fig. 2C,E; n=45 sites responding from 6 slices) and SCN region
(Fig. 2C,F; n=68 sites from 2 slices). In order to confirm that the OC-evoked changes in spike rate we
recorded represented genuine synaptically-driven responses, we then bath applied ionotropic
glutamate receptor antagonists (CNQX, 20 µM and D-AP5, 50 µM). This manipulation completely and
reversibly abolished the evoked responses at all responding channels in the pretectum, thalamus
and SCN/peri-SCN region (Fig. 2G-I; two-way RM ANOVA: Time x Treatment effects all P<0.001).
Together, these data confirm that our angled slice preparations retain a functional OT projection to
all major subcortical visual nuclei.
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Before using this model to investigate the impact of GHT signalling in more detail, however, we first
aimed to confirm that the relatively thick (600 µm) slices required would remain viable for long
enough in vitro to facilitate analysis of circadian variation in circadian network function. In fact, we
found that slice viability could be routinely maintained for longer than 24 h in vitro (Fig. 3A). Hence,
of 144 recording sites (from 5 slices) located within the SCN, 100 (~70%) displayed evidence of
circadian rhythmicity (Fig. 3B,C; see methods for analysis details). Previous work indicates that ex
vivo SCN firing rate rhythms (recorded using a wide variety of different methodologies) reliably
report phasing of the SCN in vivo and are independent of time of preparation, with peak firing
consistently occurring during the mid-late projected day (e.g. vanderLeest et al., 2009; see also
Brown & Piggins, 2007 for a detailed discussion of this point). Consistent with this earlier work, we
found that the timing of peak multiunit activity at SCN channels identified as rhythmic was strongly
clustered around the mid-late projected day (Fig. 3D).
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As expected for a major target of SCN output (Abrahamson & Moore, 2001), we also identified
electrode sites located just dorsal to the SCN (in the vSPZ) exhibiting evidence of circadian variation
in firing activity. Among this group, however, the proportion of sites identified as rhythmic was
significantly lower than for SCN located electrodes (Fig. 3C; n=61/114 vSPZ recording sites; Fisher’s
exact test P<0.01). Moreover, the timing of peak firing for rhythmic vSPZ channels was significantly
less well clustered and had a median phase significantly later than observed in the SCN (Fig. 3D;
P<0.01 and P<0.001 respectively; see methods for details of analysis).
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To further evaluate the viability of these angled slices, at the end of these long term recordings
(>26h after slice preparation) we bath applied NMDA (20μM, 5min application), a manipulation
which should drive increases in firing in most SCN neurons (~80% of cells in mice of a similar strain
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background; Cutler et al., 2003). As expected, this treatment drove robust changes in firing (see
methods) in >70% of channels located across the SCN and vSPZ (Fig. 3E,F; n=180/258 channels, no
significant difference in responses between SCN and vSPZ; Fisher’s exact test P=0.22). Collectively
then, these data establish that our preparation remains viable for greater than 1 circadian cycle in
vitro.
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Impact of GHT signals on spontaneous SCN activity
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Since our angled slice preparations retain intact OT projections across the extended circadian
system, we expected that connections between the SCN, IGL/vLGN and PON (which follow the same
trajectory) should be similarly preserved in this model. Here we specifically focused on the GHT,
although in pilot experiments (6 slices, data not shown) we were also able to readily detect
communication between the IGL/vLGN and PON.
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To test for the retention of a functional GHT, we next electrically stimulated the IGL/vLGN region via
concentric electrodes while recording from the SCN and vSPZ (Fig. 4A). Based on the predominately
GABAergic nature of IGL/vLGN neurons (Moore & Card, 1994; Morin & Blanchard, 2001), to
maximise our chances of observing inhibitory SCN responses to GHT stimulation, we performed
these recordings across the mid-late projected day when spontaneous SCN firing is highest (ZT 4-11).
In line with data obtained following in vivo stimulation of the IGL/vLGN (Roig et al., 1997; González &
Dyball, 2006), we found that single pulse stimulation of the GHT could evoke either inhibitory (Fig.
4B) or excitatory responses (Fig. 4C) across individual electrode sites within the SCN (n=21 and n=27
sites respectively from 12 slices).
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Of note, we observed differences in the latency of increases vs. decreases in SCN firing following
thalamic stimulation, with inhibitions typically displaying longer and more variable latency relative to
the usually faster excitatory responses (Fig. 4D; median±SD time to peak : 53±32 ms vs. 18±10 ms
respectively; Mann-Whitney test: P<0.001). These values represent time to peak response rather
than response onset (which is not always easy to accurately assess for inhibitory responses) and are
equivalent those reported by previous in vivo studies (Roig et al., 1997; González & Dyball, 2006).
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Since we observed a relatively low proportion of sites showing inhibitory responses to GHT
stimulation in the experiments above, in some cases, we also evaluated the possibility that single
pulse stimulation was simply insufficient to evoke a measureable inhibition in SCN firing. We found
no evidence, however, that trains of GHT stimulation (5 stimuli at 20 or 40 Hz), designed to
approximate light-driven burst firing seen in the visual thalamus (Brown et al., 2010; Howarth et al.,
2014), resulted in a greater magnitude or proportion of sites exhibiting inhibitory responses (Fig. 4E;
n=12 sites; one-way ANOVA; P>0.05).
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In keeping with previous in vivo data then, thalamic stimulation in our angled slice preparation
evokes a mixture of excitatory and inhibitory responses. While the reported excitatory actions of
GABA within the SCN (Wagner et al., 1997; Choi et al., 2008; Irwin & Allen, 2009; Farajnia et al.,
2014) could, in principle, provide an explanation for the relatively high number of electrode sites
showing excitatory responses to thalamic stimulation, our subsequent experiments rule this out as a
primary explanation. Hence, where tested (n=4 recording sites), excitatory responses were
completely abolished by bath application of ionotropic glutamate receptor blockers (Fig. 4C,G). By
contrast, whereas inhibitory responses were reliably blocked by the GABAA receptor antagonist (+)-
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bicuculline (BIC; 20µM), excitatory responses were consistently unaffected (Fig. 4F-H; n= 5 and 23
sites tested respectively).
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While the presence of a very small number of glutamatergic cells in the mouse IGL-region
(experiment: 73818754; (Lein et al., 2007)) provides one possible origin for the excitatory responses
we observed above, it is important to also note that many of the ipRGCs innervating the IGL send
axon collaterals to the SCN (Pickard, 1985; Morin et al., 2003). Accordingly, we think it more likely
that antidromic activation of this ipRGC population accounts for the excitatory responses we
observed in some SCN neurons, as described previously for increased SCN Fos-like immunoreactivity
following electrical stimulation of the thalamus (Treep et al., 1995).
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To circumvent this issue, we next turned to an optogenetics-based approach to selectively activate
GABAergic IGL/vLGN neurons in a temporally controlled manner. To this end, we employed a mouse
line (GAD2-cre; Ai32) where Cre-recombinase specifically directs ChR2/EYFP expression to GABAergic
(GAD2-expressing) neurons (Taniguchi et al., 2011). As expected, the antibody enhanced fluorescent
signal was highly enriched throughout the IGL/vLGN region (Fig. 5A,B) and other predominantly
GABAergic central nuclei (data not shown) in GAD2-Cre; Ai32 mice, indicating robust expression of
the ChR2/EYFP. Consistent with this anatomical localisation, targeted optical stimulation (Fig. 5B;
465 nm LED; ~800mW/mm2 at fibre tip, 10 ms flash) over the IGL/vLGN (Fig. 5C-E) evoked fast and
reliable bursts of spike firing from neurons across the stimulated region.
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Having established that our approach allows us to robustly activate IGL/vLGN neurons, we next
evaluated the impact of GABAergic GHT projection neuron activity on spontaneous firing within the
SCN region (Fig. 5F). In line with our data above, while in no case did we observe any excitatory
responses in the SCN following selective stimulation of GABAergic GHT cells, we did find a small
proportion of sites (n=12 sites from 4 slices) that displayed statistically significant decreases in firing
(Fig. 5G,H; see methods). The magnitude and timecourse of the resulting inhibitory responses were
broadly similar to those following electrical stimulation (mean±SEM inhibition: 43.8±5.5%; time to
peak, median±SD: 60±25 ms) and, where tested (n=6 sites), these were abolished by the bath
application of 20 µM BIC (Fig. 5I; paired t-test P<0.01), consistent with their GABAergic origin.
Together then, our data suggest that, while GABAergic GHT projections are clearly intact in our
angled slice preparation, they appear to have, at most, quite modest effects on spontaneous firing in
the SCN.
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Impact of GHT signals on SCN responses to optic input
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Insofar as our ability to detect inhibitory GHT-driven responses relies on the recipient cell to be firing
action potentials, we speculated that the modest effects observed above reflected the fact that the
primary role of this projection is to modulate responses to excitatory input from the retina. Indeed,
anatomical studies suggest that GHT projections are concentrated within retinorecipient portions of
the SCN (Mikkelsen, 1990; Moore et al., 2000; Abrahamson & Moore, 2001). To establish the
influence of GHT signals on retinohypothalamic signalling, we next monitored SCN responses to
electrical stimulation of the OC (simulating retinal input) concurrent with or following optical
activation of the GABAergic GHT projection (as in Fig. 5F). Given the relatively long latency of GHTdriven inhibitory responses, we thus expected that activation of this pathway would only be able to
modulate SCN responses if this occurred before (but not synchronous with) electrical stimulation of
the OC. Moreover, to evaluate the possibility that the functional impact of GHT signals on the OC-
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evoked response might change across the circadian day, we ran this protocol (1 OC pulse/min) over
a full 24 h recording epoch (Fig. 6A).
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We first calculated, for each independent recording site within the SCN, the average response to the
several hundred stimulus presentations across the full 24 h recording epoch. We found that, in fact,
the majority (n=45/71 sites from 3 slices) of hypothalamic recording sites at which we observed OCevoked spiking also showed a statistically significant reduction in this response when GHT
stimulation occurred prior to OC stimulation (see methods). The apparent magnitude of the effect at
these ‘GHT-responsive’ sites was relatively modest (~20% reduction) but long-lasting, being evident
for all tested delays between 50 - 500 ms (Fig. 6B; two-way RM ANOVA with Sidak’s post-tests).
Consistent with our findings above, very few of these sites showed detectable changes in
spontaneous firing following GHT stimulation (n=4/45 sites passing criteria for responsiveness, see
methods for details). Moreover, in line with anatomical studies indicating stronger GHT input to the
ipsilateral vs. contralateral SCN (Pickard, 1982; Mikkelsen, 1990; Moore et al., 2000), we also found
that the magnitude of the GHT-driven inhibition of OC-evoked responses (but not the proportion of
GHT-responsive sites) was significantly higher for sites located ipsilateral to the stimulated GHT (Fig.
6C; t-test, P<0.05).
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We also sought to determine the extent to which the multiunit responses described above reflected
the activity of individual units within the SCN. To this end we performed PCA-based spike sorting on
data from channels were we observed OC-evoked changes in firing. From these 71 channels we were
able to isolate 23 single units that exhibited clear excitatory responses to OC-stimulation. More than
half of these units (n=12/23) exhibited a significant GHT-driven modulation in the amplitude of OCevoked responses, a proportion that was not statistically different from the proportion of channels
identified as GHT responsive (Fishers exact test, P=0.24). Importantly, the majority of these GHTresponsive units (n=10/12) were isolated from electrode sites where multiunit data was similarly
modulated by GHT input, with only a few instances where single units isolated from GHT-responsive
channels lacked a significant modulation (n=3 units from 13 channels) or vice versa (n=2 units from
10 channels). Moreover, the magnitude of the GHT-driven modulations in the responding single
units were not significantly different from those we report for multiunit data (analysis as in Fig 6B;
two-way RM ANOVA; P=0.47 for population and P=0.43 for interactionxGHT stimulation latency). In
sum, these data indicate that our analysis based on multiunit responses does not lead us to
substantially underestimate the impact of GHT-inputs on individual SCN neurons.
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We next then evaluated the extent to which these GHT-driven modulations of hypothalamic
responses to optic input varied across the circadian cycle. As expected, given the nocturnal increase
in SCN sensitivity to light (Meijer et al., 1996; Meijer et al., 1998; Brown et al., 2011; van Oosterhout
et al., 2012), OC-evoked multiunit responses themselves exhibited a very pronounced circadian
variation with peak activity occurring during the early projected night, both following synchronous
activation of the GHT and following GHT pre-stimulation (Fig. 6A,D,E). Importantly, however, we
could detect clear evidence of a circadian variation in the functional impact of GHT signals (Fig. 6F;
one-way RM ANOVA of 6 h binned data; P<0.001): thus, the percentage reduction in OC-evoked
responses was ~two-fold greater during the projected day vs. the early-mid projected night (Fig.
6F).Based on these data, we next asked whether this variation in the functional impact of GHT
stimulation reflected a change in the output/excitability of GABAergic neurons in the IGL/vLGN
region, or might instead reflect an SCN intrinsic mechanism. To this end, we directly recorded OC-
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and optically-evoked responses from the IGL/vLGN region over a 24 h recording epoch (Fig. 6G,H;
n=34 sites from 3 slices). While, on average, we observed a slight decline in optically and electrically
evoked activity towards the end of these recordings (Fig. 6H), we saw no clear evidence for any
pronounced circadian variation in either spontaneous or evoked activity across the IGL/vLGN (see
methods for analysis details). In summary then, we infer that daily variation in the functional impact
of GHT signals on SCN responses involves a mechanism local to the SCN itself.
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Focusing on the projected day, where GHT signals exert the most substantial effects on SCN
response to OT input, we next sought to better understand the properties and mechanistic basis of
this influence. Hence, although by definition this mechanism is likely to involve GABA, many IGL cells
co-express NPY and/or other neuromodulators (Moore & Card, 1994; Morin & Blanchard, 1995;
Morin & Blanchard, 2001). To this end, employing similar approaches to those described above, we
investigated OC-evoked responses in the SCN following optical stimulation of GHT GABAergic cells at
variable latencies, in the presence and absence of GABA receptor blocking drugs (Fig. 7A; data
analysed by two-way RM ANOVA).
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As before, we observed a very long-lasting inhibition of OC-evoked responses at many sites (n=44/81
OC-responsive sites from 3 slices) following optical stimulation of the IGL/vLGN. This effect was
maximal at GHT pre-stimulation latencies of 50 - 100 ms and began to diminish at longer latencies
(Fig. 7B; 1s timepoint P<0.01 vs. 50 and 100ms; Sidak’s post-test). Of note, blockade of GABAA
receptors (20 µM BIC) significantly attenuated, but did not completely block, the inhibitory influence
of GHT pre-stimulation, the effect being most pronounced for short latency inhibitions (Fig. 7B). By
contrast, when we then co-applied BIC with the GABAB receptor antagonist CGP55845 (3 µM), GHTdriven inhibitory responses were almost completely abolished at all latencies (Fig. 7B).
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We also noted that blocking GABA receptor signalling produced a general enhancement in the
magnitude of OC-driven responses at these GHT-responsive channels (mean±SEM: 1.0±0.1, 1.6±0.2
and 2.5±0.3 evoked spikes at 0ms delay for baseline, BIC and BIC+CGP respectively; one-way RM
ANOVA with Sidak’s multiple comparison test, P<0.001 for baseline vs. BIC and BIC vs. BIC+CGP).
Similar results were also obtained for GHT-unresponsive channels (mean±SEM: 1.2±0.1, 2.0±0.2 and
2.9±0.3 evoked spikes for baseline, BIC and BIC+CGP respectively, P<0.001 for baseline vs. BIC and
BIC vs. BIC+CGP).
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Collectively, our data above indicate that GABA signalling is a major regulator of SCN responses to
retinal input, at least a portion of which derives from the activity of afferents from the GHT. Indeed,
the modulatory activity of the GHT revealed here seems to arise almost entirely via GABA signalling,
with GABAA receptors mediating initial components of the response and GABAB receptors driving the
longer lasting elements. We subsequently confirmed this view with an additional experiment
focusing on early components (<100ms) of the GHT-mediated inhibition (Fig. 7C; n=14 GHT
responsive sites from 3 slices). Consistent with the 20 – 30 ms latency reported by antidromic
activation of IGL cells from the SCN (Zhang & Rusak, 1989; Blasiak & Lewandowski, 2013), we
observed statistically significant reductions in OC-evoked spiking with GHT-prestimulation latencies
as low as 25 ms (two-way RM ANOVA with Sidak’s post-tests). Importantly, the inhibitory effects of
GHT-stimulation at all latencies tested here were almost completely blocked by BIC alone at all
delays tested (Fig. 7C).
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Manipulating GHT signalling modulates specific features of SCN-driven light responses
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Collectively, our data above suggest that GABAergic cells contributing to the GHT exert very little
effect on spontaneous activity across the SCN but play a more substantial role in gating SCN
responses to optic input. Based on these findings we next sought to understand the extent to which
light-driven changes in GHT output might modulate SCN responses to visual signals. Superficially,
one might assume that because the route for any potential light-driven GHT output to reach the SCN
is longer than that for direct retinal input to this region, these GHT-derived inhibitory signals would
be too slow to modulate SCN visual responses. On the other hand, visual stimuli relevant to the
circadian system would be expected to produce sustained firing in RGCs (e.g. Wong, 2012) which,
given the very persistent nature of the GHT-driven inhibition (Fig. 7), could quite substantially
modulate on-going SCN light responses. To determine then how/whether GHT activity contributes to
SCN visual signalling, we monitored light responses directly from the SCN via multi-electrode
recordings in anaesthetised mice, before and after unilateral inhibition of spiking in the visual
thalamus.
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To achieve robust inhibition of GHT signalling we performed relatively large infusions of the GABAA
receptor agonist muscimol (1 mM; 2-3 μl injection at 1 μl/min) directly into the visual thalamus while
simultaneously monitoring multiunit firing via a linear multi-electrode array attached to the drugdelivery cannula (Fig. 8A,B). This manipulation rapidly abolished spontaneous and light-driven
spiking activity at recording sites located up to 750 μm dorsal to the tip of the drug delivery cannula
(Fig. 8C). Assuming an approximately spherical spread of the drug (as observed for dye used to mark
the electrode track, Fig. 8A), we inferred then that this method of drug delivery inhibits spike firing
across the vast majority of the visual thalamus (including that of cells contributing to the GHT).
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We next turned our attention to the impact of GHT inhibition on light-evoked activity within the
SCN. Although our in vitro data suggested that GHT-driven signals exert a stronger inhibitory effect
during the projected day, we here instead chose to perform these in vivo experiments during the
early-mid projected night (ZT 12-17). Hence, across this epoch, light-driven SCN activity is more
readily quantifiable and is associated with more robust circadian phase resetting effects (Meijer et
al., 1996; Meijer et al., 1998; Brown et al., 2011; van Oosterhout et al., 2012). In fact, despite
performing these recordings at a time when the GHT exerts its most modest effects, we still
observed pronounced changes in multiunit SCN light responses following inhibition of the ipsilateral
GHT projection (Fig. 8D,G). In particular, there were substantially enhanced responses when light
pulses (405 nm, 5 s, 11.4 - 15.4 log effective photons/cm2/s) were applied to just the contralateral or
to both eyes (Fig. 8G; F-test for identical irradiance response relationship pre vs. post muscimol,
P=0.001 and P=0.002 respectively; n=63 light responsive sites from 5 mice). This effect stemmed
primarily from an increase in maximal response amplitude rather than sensitivity, since in both
cases, sigmoidal fit parameters for pre- vs. post-GHT inhibition data were identical when we
normalised those datasets to exhibit a common maximal value (F-test; P=0.999 and P=0.984; data
not shown). We did not, however, detect any statistically significant changes in the irradiance
response relationship for stimulation of the ipsilateral retina following muscimol application (Fig. 8G;
F-test; P=0.118). Together then, these data suggest that the (inhibitory) GHT projection is primarily
activated by crossed retinal inputs.
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In additional sets of experiments, we evaluated the impact of inhibiting the contralateral GHT
projection (Fig. 8E,H) and assessed the possibility that our data above may have been influenced by
spontaneous/circadian changes in SCN photic sensitivity over the tested recording epochs (Fig. 8F,I;
identical protocol to the above but with no muscimol injection). Importantly, when we did not infuse
muscimol, there was no significant change in responsiveness to any of the stimuli we employed over
the tested recording epochs (Fig. 8I; F-test; P=0.250, P=0.101 and P=0.994 respectively for changes in
irradiance response relationship to contralateral, ipsilateral and binocular light steps; n=127 light
responsive sites from 6 mice). Despite a trend towards increased SCN responses to stimulation of
the ipsilateral retina, there was a similar lack of statistically significant differences in responsiveness
following inhibition of the contralateral GHT (Fig. 8F, H; F-test; P=0.481, P=0.06, P=0.318 respectively
for contralateral, ipsilateral and binocular light steps; n=33 light responsive sites from 3 mice). These
later data thus broadly align with findings from anatomical studies (Pickard, 1982; Mikkelsen, 1990;
Moore et al., 2000) and our in vitro experiments (Fig. 6C) indicating that the contralateral GHT
projection is weaker than that arising from the ipsilateral IGL/vLGN.
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We also investigated whether inhibition of GHT signalling changed spontaneous activity within the
SCN in any way. Although we found statistically significant increases in spontaneous firing under all
conditions tested in Fig. 8D-F (mean±SEM: 123±38 vs. 45±15 and 9±3% above baseline for ipsilateral,
contralateral or no GHT inhibition respectively, paired t-tests, all P<0.05), the magnitude of this
effect was substantially greater following inhibition of the contralateral GHT (one-way ANOVA with
Dunnett’s post-hoc test, P<0.001 vs. no inhibition). It is also worth noting here that the average
change in spontaneous population activity following inhibition of the ipsilateral GHT (6.1±2.4
spikes/s) was significantly smaller than the change in light-evoked firing evoked by this manipulation
(19.6±2.4 spikes/s for 15.4 log photons/cm2/s presented to both eyes; paired t-test, P<0.001).
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Collectively, the data above suggest that, in vivo, inhibitory input from the ipsilateral GHT is active
under low light conditions and is further increased by the activity of crossed retinal projections. This
conclusion, that GHT input is primarily stimulated by crossed retinal inputs, derives from the lack of
overt changes in the irradiance response relationship for SCN responses to ipsilateral retinal
stimulation following thalamic inhibition (Fig. 8G). However, since we have previously shown that
many SCN neurons preferentially receive input from just one eye (Walmsley & Brown, 2015), an
alternate explanation for these findings is that GHT inputs preferentially converge on SCN neurons
that receive contralateral retinal input.
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To evaluate this latter possibility, we next asked whether artificially stimulating GHT activity (by
optogenetic stimulation of GABAergic neurons in the IGL/vLGN region) could adjust SCN responses
to contralateral and/or ipsilateral retinal stimulation. Accordingly, we implanted recording probes
with attached optical fibres (‘optrodes’; see methods for further details) into the IGL region of GAD2Cre;Ai32 mice, allowing us to illuminate large portions of the IGL/vLGN and to monitor the resulting
activity (Fig. 9A-C). Using this approach, we then evaluated SCN multiunit responses (Fig. 9D) to
retinal stimulation (5s steps, 15.4 log effective photons/cm2/s) in the absence or presence of trains
of optogenetic stimulation (10ms flashes at 5-20Hz for 5s).
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Since there is a delay of 30-40ms before visually evoked activity becomes first detectable within the
SCN and LGN (van Diepen et al., 2013; Howarth et al., 2014; Walmsley & Brown, 2015), we reasoned
that the first pulse of these stimulus trains should be sufficient to increase the inhibitory influence of
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the GHT prior to the arrival of any retinal input. Thus, assuming that GHT inputs do not preferentially
converge on SCN neurons receiving contralateral retinal input, we expected that initial components
of the SCN visual response should be inhibited regardless of which eye we stimulated. By contrast,
we reasoned that the effect of continued optogenetic stimulation on SCN responses should be much
greater under conditions when GHT activity is normally low (i.e. during ipsilateral visual stimulation).
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Consistent with this reasoning, we found that the earliest components of the light-evoked multiunit
firing (0-250ms after visual stimulation), were reliably reduced by concomitant optogenetic
stimulation of the GHT regardless of which eye or stimulation frequency we used (Fig. 9E,F; n=72
light responsive SCN sites from 6 mice; two-way RM ANOVA with Sidak’s post-tests). These data thus
confirm that the GHT is capable of modulating SCN responses to input from either retina. We also
found that, while optogenetic stimulation at 5 and 10Hz could also suppress later components (3-5s
after stimulus onset) of the SCN response to ipsilateral retinal inputs, there were no statistically
significant reductions in the responses to visual stimuli that also involved the contralateral retina at
any frequency tested (Fig. 9E,G; two-way RM ANOVA with Sidak’s post-tests).
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We interpret these latter data as reflecting the fact that the GHT is already much more active during
contralateral retinal stimulation and, as such, further increasing this activity with optogenetic stimuli
has negligible effects on the inhibitory signals sent to the SCN. Analysis of optogenetically-driven
changes in thalamic activity broadly supported this hypothesis (Fig. 9H,I). Here we specifically
focused on recording sites in the IGL/vLGN region where we observed both visual and
optogenetically evoked responses (n=41 sites from 6 mice). Since only a subset of IGL/vLGN neurons
contribute to the GHT, the population activity observed here is not necessarily directly reflective of
the signals being sent to the SCN. Nonetheless, while all sites exhibited very pronounced lightinduced firing during contralateral retinal stimulation, activity during stimulation of the ipsilateral
retina was negligible (and in fact, on average, slightly suppressed; Fig. 9H,I).
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Moreover, while optogenetic stimuli could significantly elevate thalamic activity spike firing during
stimulation of either or both retinas (Fig. 9H; two-way RM ANOVA with Sidak’s post-tests), the
magnitude of the resulting changes were always substantially smaller than the nominal stimulation
frequencies tested (mean±SEM = 5.1±0.4 spikes/s for 20Hz stimulation during binocular illumination
– the largest change we observed). We assume these observations reflect the fact that our
optogenetic stimuli also substantially increase local inhibition within the thalamus, limiting the
amount that we are able to increase overall firing rates. Regardless, the net result was that
optogenetic stimulation produced only a marginal increase in the already high activity when paired
with illumination of the contralateral retina but resulted in a qualitative change in (the much lower)
thalamic activity when paired with stimulation of just the ipsilateral retina (Fig. 9H,I).
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As expected based on our in vitro data, optogenetic stimulation in the absence of concomitant
retinal stimulation did not significantly alter SCN firing rates at any frequency tested (two-way RM
ANOVA with Sidak’s post-test; all P >0.05; not shown) despite producing significant, albeit modest
changes in LGN firing activity (two-way RM ANOVA with Sidak’s post-test; P >0.001 at all
frequencies; maximal change in firing = 1.5±0.3 spikes/s for 20Hz stimulation).
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In summary then, the data above support our view that the primary action of the GHT is to modulate
SCN responses to on-going retinal input and establish that, while the GHT is capable of modulating
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SCN responses to input from either eye, the primary sensory signal driving GHT output is light
detected by the contralateral retina.
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Discussion
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Here we show that GHT neurons play an important role in gating SCN neuronal responses to optic
input and actively modulates specific features of the SCN light response. Although existing data
imply that GHT activity can oppose light-induced phase resetting (Yannielli & Harrington, 2000; Lall &
Biello, 2002, 2003; Yannielli et al., 2004), to our knowledge, this is the first report that the GHT
directly modulates acute electrophysiological responses to light/retinal input. In addition, we
establish a novel in vitro model for studying network processing within the circadian system. Using
that model we show that GABA released from the GHT specifically modulates SCN responses to
retinal input with little effect on spontaneous activity. Moreover, we show that this modulatory
action exhibits a pronounced circadian variation. Collectively, then, our data (summarised in Fig. 10)
provide a mechanistic basis by which the GHT can shape temporal variations in the SCN response to
retinal input and influence both circadian photoentrainment as well as much more rapid lightdependent changes in downstream physiology (e.g. hormone secretion).
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Existing attempts to define how GHT signals act within the central clock have been hampered by the
lack of an in vitro model that allows for experimental manipulation of this pathway. As such, in vitro
studies have focused on pharmacological manipulation of NPY signalling as a proxy for GHT activity
(Shibata & Moore, 1993; Biello et al., 1997; Gribkoff et al., 1998; Yannielli & Harrington, 2000).
Although such information is useful, this approach provides at best an incomplete picture of the
influence of the GHT. Hence, not all GHT cells produce NPY (Moore & Card, 1994; Morin &
Blanchard, 2001), nor are the circumstances which trigger NPY release well understood. Similarly,
while studies using pharmacological manipulation of GABA signalling provide data consistent with
the idea that GHT-derived GABA release may modulate circadian photic phase resetting (Gillespie et
al., 1996; Gillespie et al., 1997; Novak & Albers, 2004), such approaches are unable to distinguish the
specific role of the GHT vs. the many other possible sources of GABAergic modulation. Our angled
slice model now provides a direct way to assess the mechanisms by which GHT signals influence SCN
activity.
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Here we employed two approaches to manipulate GHT signalling: electrical stimulation and targeted
optogenetic activation of GAD2-positive neurons. The former has the advantage that it should
activate any cell contributing to the GHT, regardless of neurochemical phenotype. The downside of
this approach, however, is that it carries the risk of indirectly triggering retinal input to the SCN, due
to antidromic activation of ipRGCs that send axon collaterals to the hypothalamus (Pickard, 1985;
Treep et al., 1995; Morin et al., 2003). We believe that the modest number of excitatory SCN
responses we observed following electrical stimulation of the GHT were likely driven by this route.
Hence, whereas ipRGC inputs to the SCN are glutamatergic (Gompf et al., 2015), almost all IGL/vLGN
neurons are GABAergic (Moore & Card, 1994; Harrington, 1997; Lein et al., 2007) and the excitatory
responses observed here were blocked by glutamate, but not GABA receptor antagonists.
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As such, here we instead used optogenetics to selectively activate GABAergic GHT projections. In line
with the results obtained using electrical stimulation, changes in spontaneous SCN firing were very
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rarely detectable following optogenetic activation of the GABAergic GHT projection. By contrast, an
influence of this pathway was readily apparent as a long-lasting inhibition of evoked spiking
(persisting for at least 1 s) at the majority of hypothalamic sites (~60%) responding to stimulation of
retinal afferents. In line with this in vitro data, optogenetic activation of the GHT in vivo could
modulate visual responses in the SCN but did not result in any detectable changes in spontaneous
firing. Similarly, pharmacological inhibition of the GHT in vivo produced much larger changes in lightevoked vs. spontaneous SCN activity. Collectively then, these observations indicate that our in vitro
preparation preserves a substantial proportion of the GHT intact and suggest that the principle
action of the GHT, at least under the conditions studied here, is to modulate SCN neuronal responses
to excitatory input.
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A number of features of our experimental design here are worth further discussion. Firstly, in order
to experimentally demonstrate GHT-driven modulatory effects in vitro, we stimulated the GHT
before activating retinal afferents. Here we find that GHT-driven inhibition of OT-evoked responses
becomes detectable within at least 25 ms, consistent with what one would predict from the latency
of antidromically evoked IGL spikes following hypothalamic stimulation (Zhang & Rusak, 1989;
Blasiak & Lewandowski, 2013). In the intact animal, RGC impulses will of course actually arrive in the
SCN slightly earlier than in the IGL/vLGN (latencies for visual responses ~30 vs. 40 ms: present study;
van Diepen et al., 2013; Howarth et al., 2014; Walmsley & Brown, 2015). As such, the SCN response
to a single RGC impulse should have terminated before any visually driven GHT-dependent signal
arrives. Importantly, however, visual stimuli sufficient to trigger meaningful circadian responses
must necessarily generate prolonged trains of RGC impulses. Indeed, even after many hours of
continuous light exposure, ipRGCs continue to fire at around 10 spikes/s (Wong, 2012). Accordingly,
during physiologically relevant trains of retinal input to the SCN, the GHT-driven inhibition we
demonstrate here is easily long-lasting enough (hundreds of milliseconds) to modulate SCN
responses to all but the very earliest components of the SCN response to a visual stimulus
(illustrated in schematic form in Fig. 10).
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Secondly, most of the analysis presented here derives from multiunit recordings. It is formally
possible that by combining signals from a heterogeneous population neurons, only some of which
receive GHT input, this use of multiunit data leads us to underestimate the impact of GHT signals on
the recipient neurons. Analysis of single cell responses form a subset of our recordings indicates that
such a possibility is very unlikely to result in us substantially underestimating the impact of GHT
input. Hence, there were very few instances where we could observe an effect of GHT stimulation at
the multiunit but not single unit level and, among single cells where we detected a significant
influence of GHT-input, the magnitude of the resulting responses were very similar to those we
report for multiunit data.
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By contrast, perhaps a more significant factor that could result in our in vitro data underrepresenting
the magnitude of GHT-driven inhibitory input to SCN neurons is that we focused on investigating the
effects of single-pulse GHT stimulation. It is formally possible that GHT-driven signals (arising via
GABA and/or NPY release) could summate during high frequency stimulation to produce a greater
inhibition. This possibility could explain then why we find that in vivo inhibition of GHT activity
(discussed in more detail below) produces a greater increase in visual response magnitude (~100%)
than one would predict from our in vitro data at an equivalent circadian phase (~25%).
Alternatively/additionally, it is worth noting that because the thalamic input to the SCN runs via the
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OT, the electrical stimulation used in our in vitro studies to activate retinal afferents will likely also
directly stimulate GHT terminals, potentially occluding the effect of prior optical stimulation of the
GHT.
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In line with the idea that electrical stimulation of the OC does itself trigger some baseline level of
GHT activity, we and others (Moldavan & Allen, 2013) find that GABA receptor blockade does indeed
result in a general increase in the magnitude of OC-evoked responses. On the other hand, the GHT is
far from the only source of GABA release in the SCN. Hence, facilitatory effects of GABA receptor
blockade on OC-evoked responses do not necessarily confirm an endogenous GHT-contribution of
this kind. In this regard, it is noteworthy that the reductions in early visual responses we observe
following optical activation of the GHT in vivo (where we expect one optogenetically-driven GHT
impulse to reach the SCN before any light-driven GHT signals do) are broadly similar in magnitude to
those we report in vitro at an equivalent circadian phase.
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On balance then, although our present work leaves these latter issues somewhat unresolved, the
inhibitory actions of GHT-stimulation we report in vitro should perhaps be considered a conservative
estimate of the extent to which these inputs regulate SCN activity. Despite this caveat, it is
abundantly clear from both our in vitro and in vivo data that GHT-derived signals exert a much more
robust effect on SCN responses to retinal input than on spontaneous firing. Such an arrangement
may, in principle arise simply because the low spontaneous activity of SCN neurons ensures that the
functional impact of GHT-dependent inhibitory signals is hard to detect in the absence of a driving
input. We think it most unlikely that this constitutes the primary explanation for the apparent lack of
effect of GHT-stimulation on spontaneous firing. Indeed the design of these experiments (where we
evaluated responses to hundreds of stimulus repeats) was specifically tailored to enable us to detect
inhibitory responses even when spontaneous activity was relatively low.
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An alternative explanation then is that GHT inputs might be arranged such that they can specifically
gate SCN responses to retinal signals without exerting major effects on spontaneous firing. In this
regard, NPY cells reportedly form axodendritic contacts with SCN neurons (Pelletier, 1990).
Moreover, it has previously been demonstrated that retinal and GABAergic terminals (potentially
including those originating from the GHT) are typically found in very close proximity within the SCN,
often converging onto the same postsynaptic dendrite (Castel et al., 1993). Since spontaneous SCN
firing is primarily generated within the soma, such an arrangement should indeed allow GABA
released by this population of GHT cells to preferentially gate responses to excitatory input. In
addition, the presence of GABAB receptors almost exclusively located on (nearby) retinal terminals
leaves open the possibility that GABA spillover might also presynaptically modulate retinal input
(Jiang et al., 1995; Moldavan et al., 2006; Belenky et al., 2008). In any of the above situations,
however, the net effect is that the functional impacts of GHT signals are most clearly evident in the
face of on-going retinal transmission.
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It is worth noting here that pharmacological activation of NPY receptors is able to suppress
spontaneous firing in the SCN (Gribkoff et al., 1998). By contrast, we found that inhibitory responses
to optogenetic activation of the GHT could be almost entirely blocked by GABA receptor antagonists.
It appears then that, under our experimental conditions, NPY released from the GHT does not exert
a major effect. In principle, it is formally possible that the approaches we used preferentially activate
non-NPY cells. This seems unlikely, however, since almost all GABAergic neurons produce GAD2
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(Zhao et al., 2013), expression of which is itself very reliably reported by the GAD2-Cre line
(Taniguchi et al., 2011). Certainly our data indicate very robust ChR2/EYFP expression in the
IGL/vLGN region. Instead then, we suggest that specific patterns of presynaptic terminal activity may
be required to evoke endogenous NPY release, with the short bursts of firing evoked by our test
stimuli primarily acting via triggering GABA release.
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Significantly, here we identify a pronounced rhythm in the functional impact of this GHT-derived
GABA release, with ~two-fold greater inhibition of OC-evoked spiking during the mid-late projected
day vs. the early projected night. As discussed below then, these observations provide a potential
mechanism by which signals from the GHT could contribute to shaping the characteristic temporal
gating of the SCN light response/clock resetting (Meijer et al., 1996; Meijer et al., 1998; Brown et al.,
2011; van Oosterhout et al., 2012). The effect we observe in vitro is not, however, associated with
any appreciable change in responses recorded directly from the IGL/vLGN region. Accordingly, we
interpret these data as reflecting a circadian variation in the impact of GHT-derived GABA release at
the level of the SCN neurons and/or retinal terminals. In line with this latter view, one previous
report does indeed indicate that GABAB receptor mediated presynaptic inhibition of
retinohypothalamic transmission is enhanced during the projected day (Moldavan & Allen, 2013).
We should note, however, that it is formally possible that GHT-derived GABA release might exhibit
circadian variation even though IGL/vLGN firing does not itself change over the circadian day.
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In interpreting the functional significance of the in vitro data discussed above, a key area to consider
is the nature of the sensory signals that actually activate GHT transmission. Owing to the challenging
nature of directly identifying GHT cells in electrophysiological recordings, existing attempts to
address this deficit have not provided a clear answer (Harrington & Rusak, 1989; Zhang & Rusak,
1989; Thankachan & Rusak, 2005; Blasiak & Lewandowski, 2013). Although we do not directly
investigate this question here, our in vivo experiments employing thalamic inhibition clearly indicate
that, during visual stimulation, activity of the ipsilateral GHT reduces SCN population responses
specifically to stimuli that target the contralateral retina.
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Due to the extensive interconnections among the SCN, IGL/vLGN and pretectum (Morin & Allen,
2006), thalamic inhibition could influence SCN light responses via various routes. Nonetheless, the
simplest interpretation of our data is that the inhibitory GABAergic neurons that form the GHT
themselves primarily receive ON-excitatory input from crossed retinal projections. Consistent with
this view, we rule out one important alternative explanation for the above data (that the GHT
preferentially impinges on SCN neurons receiving contralateral retinal input; Walmsley & Brown,
2015). Hence, we show that optogenetic activation of the GHT in vivo is capable of reducing early
components (first 250ms) of the SCN response to both ipsilateral and contralateral input. We also
show that maintained optogenetic stimulation has negligible effects on later components of the SCN
response to contralateral retinal stimulation, as expected if endogenous activity of the GHT is
already high. By contrast, maintained optogenetic stimulation at 5 and 10Hz produced continued
inhibition of responses driven by the ipsilateral retina, consistent with a much lower level of
endogenous GHT activity under these conditions.
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Regarding interpretation of this latter element of our data, we should note that at a higher
optogenetic stimulation frequency (20Hz) this inhibition of on-going ipsilateral retinal-driven activity
disappeared. This is despite the fact that our analysis of population activity in the IGL/vLGN region
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during the same recordings revealed significantly increased spiking activity (of a broadly similar
magnitude to that observed at lower frequencies). Since only a subset of neurons in the IGL/vLGN
contribute to the GHT, we assume then that the thalamic data we record here is not entirely
representative of the activity of GHT neurons. Alternatively, it is formally possible that the specific
spiking pattern of GHT afferents is also an important determinant of the overall impact on the SCN.
In either case, our general conclusion that GHT signalling is primarily activated by the illumination of
the contralateral retina is certainly supported by our observations of light evoked activity in the
IGL/vLGN (present study and previous large scale recordings of sensory responses in this region:
Howarth et al., 2014), which indicate that excitatory responses to stimulation of the ipsilateral retina
are rare (present in only ~25% of cells).
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Of note, the enhancement of SCN visual responses we observed following thalamic inhibition
showed no signs of diminishing across the duration of the visual stimulations tested here. Again, this
is consistent with both the long-lasting effects of GHT activity we observe in vitro and the sustained
visual responses driven by ipRGCs which provide a major source of input to the IGL/vLGN (Brown et
al., 2010; Wong, 2012). As such, it seems that the GHT modulates the irradiance coding properties of
SCN neurons. Based on the relationship between SCN firing and irradiance, the magnitude of this
effect is large for relatively bright stimuli (>1014 photons/cm2/s; equivalent to those encountered
around sunrise/sunset). Indeed, for contralateral light steps, we found that ~10-fold lower light
levels were required to produce SCN population firing responses equivalent to those attained before
inhibition of the GHT.
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Most significantly, the in vivo recordings discussed above occurred across a time epoch when the
GHT was expected to exert its weakest effects. Indeed, assuming a two-fold greater effect of the
GHT input during the day (as predicted by our in vitro data), it seems that input from the GHT could
be sufficient to account for a substantial component of the circadian modulation in light-driven SCN
activity observed previously (3-4-fold variation in magnitude at submaximal intensity; Meijer et al.,
1998; Brown et al., 2011; van Oosterhout et al. 2012). An intriguing question then is to what extent
does the GHT helps to define the phase response curve for behavioural photoentrainment?
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Certainly, based on data from IGL-lesioned hamsters, the GHT is not an absolute requirement for
photoentrainment under most circumstances (Harrington & Rusak, 1986; Pickard et al., 1987;
Johnson et al., 1989; Maywood et al., 1997; Edelstein & Amir, 1999a, b; Morin & Pace, 2002; Muscat
& Morin, 2006; Evans et al., 2012). Nonetheless, we consider it noteworthy that, in mice,
behavioural phase resetting responses to light are largest during the first half of the projected night
(Daan & Pittendrigh, 1976), a portion of the day when the inhibitory effects of GHT-activation are at
their lowest ebb. Insofar as we have previously shown that the magnitude of light-evoked SCN firing
is predictive of mouse circadian phase resetting responses (Brown et al., 2011), our data are thus
consistent with the possibility that GHT inputs contribute to reducing circadian responses to light
around dawn and across the projected day.
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In summary, then, our data provide new insight into the contributions of the GHT to visual
processing within the SCN. We show that the GHT actively modulates SCN responses to retinal input
and demonstrate a circadian variation in the magnitude of this effect which is consistent with an
important role in the temporal gating of photic responses, a fundamental property of the circadian
system. We also establish a new model system for studying geniculohypothalamic communication,
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which we expect to have widespread utility for many further investigations of how network
interactions across the extended circadian system contribute to daily rhythms in physiology and
behaviour.
1023
Additional information
1024
Competing interests
1025
None declared.
1026
Author contributions
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T.M.B., L.H. and L.W. designed the experiments, L.H., L.W., A.P., M.H. and T.M.B. collected and
analysed the data, T.M.B. and L.H. wrote the manuscript. All authors approved the final version of
the manuscript, agree to be accountable for all aspects of the work and all persons designated as
authors qualify for authorship. All experiments were performed at the University of Manchester, UK.
1031
Funding
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This work was funded by a Biotechnology and Biological Sciences Research Council (BBSRC, UK)
David Philips Fellowship to T.M.B. (BB/I017836/1). L.W. was funded by a BBSRC doctoral training
grant and strategic skills award.
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References
1037
1038
Abrahamson EE & Moore RY. (2001). Suprachiasmatic nucleus in the mouse: retinal innervation,
intrinsic organization and efferent projections. Brain Res 916, 172-191.
1039
1040
1041
1042
Belenky MA, Yarom Y & Pickard GE. (2008). Heterogeneous expression of gamma-aminobutyric acid
and gamma-aminobutyric acid-associated receptors and transporters in the rat
suprachiasmatic nucleus. J Comp Neurol 506, 708-732.
1043
1044
1045
Biello SM, Golombek DA & Harrington ME. (1997). Neuropeptide Y and glutamate block each other's
phase shifts in the suprachiasmatic nucleus in vitro. Neuroscience 77, 1049-1057.
1046
1047
1048
1049
Blasiak T & Lewandowski MH. (2013). Differential firing pattern and response to lighting conditions
of rat intergeniculate leaflet neurons projecting to suprachiasmatic nucleus or contralateral
intergeniculate leaflet. Neuroscience 228, 315-324.
1050
1051
1052
1053
Brown TM, Banks JR & Piggins HD. (2006). A novel suction electrode recording technique for
monitoring circadian rhythms in single and multiunit discharge from brain slices. J Neurosci
Methods 156, 173-181.
1054
1055
1056
1057
Brown TM, Gias C, Hatori M, Keding SR, Semo M, Coffey PJ, Gigg J, Piggins HD, Panda S & Lucas RJ.
(2010). Melanopsin contributions to irradiance coding in the thalamo-cortical visual system.
PLoS Biol 8, e1000558.
1058
1059
1060
Brown TM, McLachlan E & Piggins HD. (2008). Angiotensin II regulates the activity of mouse
suprachiasmatic nuclei neurons. Neuroscience 154, 839-847.
1061
1062
1063
Brown TM & Piggins HD. (2007). Electrophysiology of the suprachiasmatic circadian clock. Prog
Neurobiol 82, 229-255.
1064
1065
1066
Brown TM & Piggins HD. (2009). Spatiotemporal heterogeneity in the electrical activity of
suprachiasmatic nuclei neurons and their response to photoperiod. J Biol Rhythms 24, 44-54.
1067
1068
1069
Brown TM, Wynne J, Piggins HD & Lucas RJ. (2011). Multiple hypothalamic cell populations encoding
distinct visual information. J Physiol 589, 1173-1194.
1070
1071
1072
1073
1074
1075
Castel M, Belenky M, Cohen S, Ottersen OP & Storm-Mathisen J. (1993). Glutamate-like
immunoreactivity in retinal terminals of the mouse suprachiasmatic nucleus. Eur J Neurosci.
5, 368-381.
Chen SK, Badea TC & Hattar S. (2011). Photoentrainment and pupillary light reflex are mediated by
distinct populations of ipRGCs. Nature 476, 92-95.
1076
1077
1078
1079
Choi HJ, Lee CJ, Schroeder A, Kim YS, Jung SH, Kim JS, Kim dY, Son EJ, Han HC, Hong SK, Colwell CS &
Kim YI. (2008). Excitatory actions of GABA in the suprachiasmatic nucleus. J Neurosci 28,
5450-5459.
1080
1081
1082
1083
Cutler DJ, Haraura M, Reed HE, Shen S, Sheward WJ, Morrison CF, Marston HM, Harmar AJ & Piggins
HD. (2003). The mouse VPAC2 receptor confers suprachiasmatic nuclei cellular rhythmicity
and responsiveness to vasoactive intestinal polypeptide in vitro. Eur J Neurosci 17, 197-204.
1084
1085
1086
1087
1088
Daan, S & Pittendrigh, C. (1976). A Functional Analysis of Circadian Pacemakers in Nocturnal
Rodents: II. The Variability of Phase Response Curves. J Comp Physiol 106, 253-266.
Edelstein K & Amir S. (1999a). The intergeniculate leaflet does not mediate the disruptive effects of
constant light on circadian rhythms in the rat. Neuroscience 90, 1093-1101.
1089
1090
1091
Edelstein K & Amir S. (1999b). The role of the intergeniculate leaflet in entrainment of circadian
rhythms to a skeleton photoperiod. J Neurosci 19, 372-380.
1092
1093
1094
1095
Evans JA, Carter SN, Freeman DA & Gorman MR. (2012). Dim nighttime illumination alters
photoperiodic responses of hamsters through the intergeniculate leaflet and other photic
pathways. Neuroscience 202, 300-308.
1096
1097
1098
Farajnia S, van Westering TL, Meijer JH & Michel S. (2014). Seasonal induction of GABAergic
excitation in the central mammalian clock. Proc Natl Acad Sci U S A 111, 9627-9632.
1099
1100
1101
1102
Gillespie CF, Huhman KL, Babagbemi TO & Albers HE. (1996). Bicuculline increases and muscimol
reduces the phase-delaying effects of light and VIP/PHI/GRP in the suprachiasmatic region. J
Biol Rhythms 11, 137-144.
1103
1104
1105
1106
Gillespie CF, Mintz EM, Marvel CL, Huhman KL & Albers HE. (1997). GABA(A) and GABA(B) agonists
and antagonists alter the phase-shifting effects of light when microinjected into the
suprachiasmatic region. Brain Res 759, 181-189.
1107
1108
1109
Gompf HS, Fuller PM, Hattar S, Saper CB & Lu J. (2015). Impaired circadian photosensitivity in mice
lacking glutamate transmission from retinal melanopsin cells. J Biol Rhythms 30, 35-41.
1110
1111
1112
González JA & Dyball RE. (2006). Pinealectomy reduces optic nerve but not intergeniculate leaflet
input to the suprachiasmatic nucleus at night. J Neuroendocrinol 18, 146-153.
1113
1114
1115
Govardovskii VI, Fyhrquist N, Reuter T, Kuzmin DG & Donner K. (2000). In search of the visual
pigment template. Vis Neurosci 17, 509-528.
1116
1117
1118
Green DJ & Gillette R. (1982). Circadian rhythm of firing rate recorded from single cells in the rat
suprachiasmatic brain slice. Brain Res 245, 198-200.
1119
1120
1121
1122
Gribkoff VK, Pieschl RL, Wisialowski TA, van den Pol AN & Yocca FD. (1998). Phase shifting of
circadian rhythms and depression of neuronal activity in the rat suprachiasmatic nucleus by
neuropeptide Y: mediation by different receptor subtypes. J Neurosci 18, 3014-3022.
1123
1124
1125
Groos G & Hendriks J. (1982). Circadian rhythms in electrical discharge of rat suprachiasmatic
neurones recorded in vitro. Neurosci Lett 34, 283-288.
1126
1127
1128
1129
Güler AD, Altimus CM, Ecker JL & Hattar S. (2007). Multiple photoreceptors contribute to nonimageforming visual functions predominantly through melanopsin-containing retinal ganglion
cells. Cold Spring Harb Symp Quant Biol 72, 509-515.
1130
1131
1132
1133
Harrington ME. (1997). The ventral lateral geniculate nucleus and the intergeniculate leaflet:
interrelated structures in the visual and circadian systems. Neurosci Biobehav Rev 21, 705727.
1134
1135
1136
Harrington ME & Rusak B. (1986). Lesions of the thalamic intergeniculate leaflet alter hamster
circadian rhythms. J Biol Rhythms 1, 309-325.
1137
1138
1139
Harrington ME & Rusak B. (1989). Photic responses of geniculo-hypothalamic tract neurons in the
Syrian hamster. Vis Neurosci 2, 367-375.
1140
1141
1142
Hattar S, Kumar M, Park A, Tong P, Tung J, Yau KW & Berson DM. (2006). Central projections of
melanopsin-expressing retinal ganglion cells in the mouse. J Comp Neurol 497, 326-349.
1143
1144
1145
1146
1147
Horvath TL. (1998). An alternate pathway for visual signal integration into the hypothalamo-pituitary
axis: retinorecipient intergeniculate neurons project to various regions of the hypothalamus
and innervate neuroendocrine cells including those producing dopamine. J Neurosci 18,
1546-1558.
1148
1149
1150
Howarth M, Walmsley L & Brown TM. (2014). Binocular integration in the mouse lateral geniculate
nuclei. Curr Biol 24, 1241-1247.
1151
1152
1153
Huhman KL & Albers HE. (1994). Neuropeptide Y microinjected into the suprachiasmatic region
phase shifts circadian rhythms in constant darkness. Peptides 15, 1475-1478.
1154
1155
1156
Huhman KL, Gillespie CF, Marvel CL & Albers HE. (1996). Neuropeptide Y phase shifts circadian
rhythms in vivo via a Y2 receptor. Neuroreport 7, 1249-1252.
1157
1158
1159
1160
Huhman KL, Marvel CL, Gillespie CF, Mintz EM & Albers HE. (1997). Tetrodotoxin blocks NPY-induced
but not muscimol-induced phase advances of wheel-running activity in Syrian hamsters.
Brain Res 772, 176-180.
1161
1162
1163
Irwin RP & Allen CN. (2009). GABAergic signaling induces divergent neuronal Ca2+ responses in the
suprachiasmatic nucleus network. Eur J Neurosci 30, 1462-1475.
1164
1165
1166
Itri JN, Michel S, Vansteensel MJ, Meijer JH & Colwell CS. (2005). Fast delayed rectifier potassium
current is required for circadian neural activity. Nat Neurosci 8, 650-656.
1167
1168
1169
Jacobs GH & Williams GA. (2007). Contributions of the mouse UV photopigment to the ERG and to
vision. Doc Ophthalmol 115, 137-144.
1170
1171
1172
Janik D & Mrosovsky N. (1994). Intergeniculate leaflet lesions and behaviorally-induced shifts of
circadian rhythms. Brain Res 651, 174-182.
1173
1174
1175
Jiang ZG, Allen CN & North RA. (1995). Presynaptic inhibition by baclofen of retinohypothalamic
excitatory synaptic transmission in rat suprachiasmatic nucleus. Neuroscience 64, 813-819.
1176
1177
1178
Johnson RF, Moore RY & Morin LP. (1989). Lateral geniculate lesions alter circadian activity rhythms
in the hamster. Brain Res Bull 22, 411-422.
1179
1180
1181
Johnson RF, Smale L, Moore RY & Morin LP. (1988). Lateral geniculate lesions block circadian phaseshift responses to a benzodiazepine. Proc Natl Acad Sci U S A 85, 5301-5304.
1182
1183
1184
Kalsbeek A, Teclemariam-Mesbah R & Pévet P. (1993). Efferent projections of the suprachiasmatic
nucleus in the golden hamster (Mesocricetus auratus). J Comp Neurol 332, 293-314.
1185
1186
1187
Kubota A, Inouye ST & Kawamura H. (1981). Reversal of multiunit activity within and outside the
suprachiasmatic nucleus in the rat. Neurosci Lett 27, 303-308.
1188
1189
1190
Lall GS & Biello SM. (2002). Attenuation of phase shifts to light by activity or neuropeptide Y: a time
course study. Brain Res 957, 109-116.
1191
1192
1193
Lall GS & Biello SM. (2003). Attenuation of circadian light induced phase advances and delays by
neuropeptide Y and a neuropeptide Y Y1/Y5 receptor agonist. Neuroscience 119, 611-618.
1194
1195
1196
1197
1198
1199
1200
1201
1202
1203
1204
1205
1206
1207
1208
Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard A, Boe AF, Boguski MS, Brockway KS,
Byrnes EJ, Chen L, Chen TM, Chin MC, Chong J, Crook BE, Czaplinska A, Dang CN, Datta S, Dee
NR, Desaki AL, Desta T, Diep E, Dolbeare TA, Donelan MJ, Dong HW, Dougherty JG, Duncan
BJ, Ebbert AJ, Eichele G, Estin LK, Faber C, Facer BA, Fields R, Fischer SR, Fliss TP, Frensley C,
Gates SN, Glattfelder KJ, Halverson KR, Hart MR, Hohmann JG, Howell MP, Jeung DP,
Johnson RA, Karr PT, Kawal R, Kidney JM, Knapik RH, Kuan CL, Lake JH, Laramee AR, Larsen
KD, Lau C, Lemon TA, Liang AJ, Liu Y, Luong LT, Michaels J, Morgan JJ, Morgan RJ, Mortrud
MT, Mosqueda NF, Ng LL, Ng R, Orta GJ, Overly CC, Pak TH, Parry SE, Pathak SD, Pearson OC,
Puchalski RB, Riley ZL, Rockett HR, Rowland SA, Royall JJ, Ruiz MJ, Sarno NR, Schaffnit K,
Shapovalova NV, Sivisay T, Slaughterbeck CR, Smith SC, Smith KA, Smith BI, Sodt AJ, Stewart
NN, Stumpf KR, Sunkin SM, Sutram M, Tam A, Teemer CD, Thaller C, Thompson CL, Varnam
LR, Visel A, Whitlock RM, Wohnoutka PE, Wolkey CK, Wong VY, Wood M, Yaylaoglu MB,
Young RC, Youngstrom BL, Yuan XF, Zhang B, Zwingman TA & Jones AR. (2007). Genomewide atlas of gene expression in the adult mouse brain. Nature 445, 168-176.
1209
1210
1211
Long MA, Jutras MJ, Connors BW & Burwell RD. (2005). Electrical synapses coordinate activity in the
suprachiasmatic nucleus. Nat Neurosci 8, 61-66.
1212
1213
1214
1215
1216
Madisen L, Mao T, Koch H, Zhuo JM, Berenyi A, Fujisawa S, Hsu YW, Garcia AJ, Gu X, Zanella S, Kidney
J, Gu H, Mao Y, Hooks BM, Boyden ES, Buzsáki G, Ramirez JM, Jones AR, Svoboda K, Han X,
Turner EE & Zeng H. (2012). A toolbox of Cre-dependent optogenetic transgenic mice for
light-induced activation and silencing. Nat Neurosci 15, 793-802.
1217
1218
1219
1220
Marchant EG, Watson NV & Mistlberger RE. (1997). Both neuropeptide Y and serotonin are
necessary for entrainment of circadian rhythms in mice by daily treadmill running schedules.
J Neurosci 17, 7974-7987.
1221
1222
1223
1224
Maywood ES, Smith E, Hall SJ & Hastings MH. (1997). A thalamic contribution to arousal-induced,
non-photic entrainment of the circadian clock of the Syrian hamster. Eur J Neurosci 9, 17391747.
1225
1226
1227
Meijer JH, Watanabe K, Détàri L & Schaap J. (1996). Circadian rhythm in light response in
suprachiasmatic nucleus neurons of freely moving rats. Brain Res 741, 352-355.
1228
1229
1230
1231
Meijer JH, Watanabe K, Schaap J, Albus H & Détári L. (1998). Light responsiveness of the
suprachiasmatic nucleus: long-term multiunit and single-unit recordings in freely moving
rats. J Neurosci 18, 9078-9087.
1232
1233
1234
1235
Mikkelsen JD. (1990). Projections from the lateral geniculate nucleus to the hypothalamus of the
Mongolian gerbil (Meriones unguiculatus): an anterograde and retrograde tracing study. J
Comp Neurol 299, 493-508.
1236
1237
1238
Mikkelsen JD. (1992). The organization of the crossed geniculogeniculate pathway of the rat: a
Phaseolus vulgaris-leucoagglutinin study. Neuroscience 48, 953-962.
1239
1240
1241
1242
Moldavan MG & Allen CN. (2013). GABAB receptor-mediated frequency-dependent and circadian
changes in synaptic plasticity modulate retinal input to the suprachiasmatic nucleus. J
Physiol 591, 2475-2490.
1243
1244
1245
1246
Moldavan MG, Irwin RP & Allen CN. (2006). Presynaptic GABA(B) receptors regulate
retinohypothalamic tract synaptic transmission by inhibiting voltage-gated Ca2+ channels. J
Neurophysiol 95, 3727-3741.
1247
1248
1249
Moore RY & Card JP. (1994). Intergeniculate leaflet: an anatomically and functionally distinct
subdivision of the lateral geniculate complex. J Comp Neurol 344, 403-430.
1250
1251
1252
Moore RY, Weis R & Moga MM. (2000). Efferent projections of the intergeniculate leaflet and the
ventral lateral geniculate nucleus in the rat. J Comp Neurol 420, 398-418.
1253
1254
Morin LP & Allen CN. (2006). The circadian visual system, 2005. Brain Res Rev 51, 1-60.
1255
1256
1257
1258
Morin LP & Blanchard J. (1995). Organization of the hamster intergeniculate leaflet: NPY and ENK
projections to the suprachiasmatic nucleus, intergeniculate leaflet and posterior limitans
nucleus. Vis Neurosci 12, 57-67.
1259
1260
1261
1262
Morin LP & Blanchard JH. (2001). Neuromodulator content of hamster intergeniculate leaflet
neurons and their projection to the suprachiasmatic nucleus or visual midbrain. J Comp
Neurol 437, 79-90.
1263
1264
1265
1266
Morin LP, Blanchard JH & Provencio I. (2003). Retinal ganglion cell projections to the hamster
suprachiasmatic nucleus, intergeniculate leaflet, and visual midbrain: bifurcation and
melanopsin immunoreactivity. J Comp Neurol 465, 401-416.
1267
1268
1269
1270
Morin LP, Goodless-Sanchez N, Smale L & Moore RY. (1994). Projections of the suprachiasmatic
nuclei, subparaventricular zone and retrochiasmatic area in the golden hamster.
Neuroscience 61, 391-410.
1271
1272
1273
Morin LP & Pace L. (2002). The intergeniculate leaflet, but not the visual midbrain, mediates hamster
circadian rhythm response to constant light. J Biol Rhythms 17, 217-226.
1274
1275
1276
1277
Mrugala M, Zlomanczuk P, Jagota A & Schwartz WJ. (2000). Rhythmic multiunit neural activity in
slices of hamster suprachiasmatic nucleus reflect prior photoperiod. Am J Physiol Regul
Integr Comp Physiol 278, R987-994.
1278
1279
1280
Muscat L & Morin LP. (2006). Intergeniculate leaflet: contributions to photic and non-photic
responsiveness of the hamster circadian system. Neuroscience 140, 305-320.
1281
1282
1283
Nakamura W, Yamazaki S, Nakamura TJ, Shirakawa T, Block GD & Takumi T. (2008). In vivo
monitoring of circadian timing in freely moving mice. Curr Biol 18, 381-385.
1284
1285
1286
Novak CM & Albers HE. (2004). Circadian phase alteration by GABA and light differs in diurnal and
nocturnal rodents during the day. Behav Neurosci 118, 498-504.
1287
1288
1289
Novak CM, Ehlen JC, Huhman KL & Albers HE. (2004). GABA(B) receptor activation in the
suprachiasmatic nucleus of diurnal and nocturnal rodents. Brain Res Bull 63, 531-535.
1290
1291
1292
1293
Nygård M, Hill RH, Wikström MA & Kristensson K. (2005). Age-related changes in
electrophysiological properties of the mouse suprachiasmatic nucleus in vitro. Brain Res Bull
65, 149-154.
1294
1295
1296
Paxinos G & Franklin K. (2001). The Mouse Brain in Stereotaxic Coordinates, Second Edition.
Academic Press, San Diego.
1297
1298
1299
Pelletier G. (1990). Ultrastructural localization of neuropeptide Y in the hypothalamus. Ann N Y Acad
Sci 611, 232-246.
1300
1301
1302
Pennartz CM, de Jeu MT, Bos NP, Schaap J & Geurtsen AM. (2002). Diurnal modulation of pacemaker
potentials and calcium current in the mammalian circadian clock. Nature 416, 286-290.
1303
1304
1305
Pickard GE. (1982). The afferent connections of the suprachiasmatic nucleus of the golden hamster
with emphasis on the retinohypothalamic projection. J Comp Neurol 211, 65-83.
1306
1307
1308
1309
Pickard GE. (1985). Bifurcating axons of retinal ganglion cells terminate in the hypothalamic
suprachiasmatic nucleus and the intergeniculate leaflet of the thalamus. Neurosci Lett 55,
211-217.
1310
1311
1312
Pickard GE, Ralph MR & Menaker M. (1987). The intergeniculate leaflet partially mediates effects of
light on circadian rhythms. J Biol Rhythms 2, 35-56.
1313
1314
1315
1316
Reinhard K, Tikidji-Hamburyan A, Seitter H, Idrees S, Mutter M, Benkner B & Münch TA. (2014). Stepby-step instructions for retina recordings with perforated multi electrode arrays. PLoS One 9,
e106148.
1317
1318
1319
1320
Roig JA, Granados-Fuentes D & Aguilar-Roblero R. (1997). Neuronal subpopulations in the
suprachiasmatic nuclei based on their response to retinal and intergeniculate leaflet
stimulation. Neuroreport 8, 885-889.
1321
1322
1323
Rusak B, Meijer JH & Harrington ME. (1989). Hamster circadian rhythms are phase-shifted by
electrical stimulation of the geniculo-hypothalamic tract. Brain Res 493, 283-291.
1324
1325
1326
1327
Sakhi K, Wegner S, Belle MD, Howarth M, Delagrange P, Brown TM & Piggins HD. (2014). Intrinsic
and extrinsic cues regulate the daily profile of mouse lateral habenula neuronal activity. J
Physiol 592, 5025-5045.
1328
1329
1330
1331
Schaap J, Albus H, VanderLeest HT, Eilers PH, Détári L & Meijer JH. (2003). Heterogeneity of rhythmic
suprachiasmatic nucleus neurons: Implications for circadian waveform and photoperiodic
encoding. Proc Natl Acad Sci U S A 100, 15994-15999.
1332
1333
1334
1335
Schaap J, Bos NP, de Jeu MT, Geurtsen AM, Meijer JH & Pennartz CM. (1999). Neurons of the rat
suprachiasmatic nucleus show a circadian rhythm in membrane properties that is lost during
prolonged whole-cell recording. Brain Res 815, 154-166.
1336
1337
1338
Shibata S & Moore RY. (1993). Neuropeptide Y and optic chiasm stimulation affect suprachiasmatic
nucleus circadian function in vitro. Brain Res 615, 95-100.
1339
1340
1341
Shibata S, Oomura Y, Kita H & Hattori K. (1982). Circadian rhythmic changes of neuronal activity in
the suprachiasmatic nucleus of the rat hypothalamic slice. Brain Res 247, 154-158.
1342
1343
1344
Smith RD, Inouye S & Turek FW. (1989). Central administration of muscimol phase-shifts the
mammalian circadian clock. J Comp Physiol A 164, 805-814.
1345
1346
1347
1348
Taniguchi H, He M, Wu P, Kim S, Paik R, Sugino K, Kvitsiani D, Kvitsani D, Fu Y, Lu J, Lin Y, Miyoshi G,
Shima Y, Fishell G, Nelson SB & Huang ZJ. (2011). A resource of Cre driver lines for genetic
targeting of GABAergic neurons in cerebral cortex. Neuron 71, 995-1013.
1349
1350
1351
1352
Tcheng TK & Gillette MU. (1996). A novel carbon fiber bundle microelectrode and modified brain
slice chamber for recording long-term multiunit activity from brain slices. J Neurosci
Methods 69, 163-169.
1353
1354
1355
Thankachan S & Rusak B. (2005). Juxtacellular recording/labeling analysis of physiological and
anatomical characteristics of rat intergeniculate leaflet neurons. J Neurosci 25, 9195-9204.
1356
1357
1358
Tousson E & Meissl H. (2004). Suprachiasmatic nuclei grafts restore the circadian rhythm in the
paraventricular nucleus of the hypothalamus. J Neurosci 24, 2983-2988.
1359
1360
1361
Treep JA, Abe H, Rusak B & Goguen DM. (1995). Two distinct retinal projections to the hamster
suprachiasmatic nucleus. J Biol Rhythms 10, 299-307.
1362
1363
1364
van Diepen HC, Ramkisoensing A, Peirson SN, Foster RG & Meijer JH. (2013). Irradiance encoding in
the suprachiasmatic nuclei by rod and cone photoreceptors. FASEB J 27, 4204-4212.
1365
1366
1367
1368
van Oosterhout F, Fisher SP, van Diepen HC, Watson TS, Houben T, VanderLeest HT, Thompson S,
Peirson SN, Foster RG & Meijer JH. (2012). Ultraviolet light provides a major input to nonimage-forming light detection in mice. Curr Biol 22, 1397-1402.
1369
1370
1371
VanderLeest HT, Houben T, Michel S, Deboer T, Albus H, Vansteensel MJ, Block GD & Meijer JH.
(2007). Seasonal encoding by the circadian pacemaker of the SCN. Curr Biol 17, 468-473.
1372
1373
1374
1375
vanderLeest HT, Vansteensel MJ, Duindam H, Michel S & Meijer JH. (2009). Phase of the electrical
activity rhythm in the SCN in vitro not influenced by preparation time. Chronobiol Int 26,
1075-1089.
1376
1377
1378
Wagner S, Castel M, Gainer H & Yarom Y. (1997). GABA in the mammalian suprachiasmatic nucleus
and its role in diurnal rhythmicity. Nature 387, 598-603.
1379
1380
1381
Walmsley L & Brown TM. (2015). Eye-specific visual processing in the mouse suprachiasmatic nuclei.
J Physiol 593, 1731-1743.
1382
1383
1384
1385
Walmsley L, Hanna L, Mouland J, Martial F, West A, Smedley AR, Bechtold DA, Webb AR, Lucas RJ &
Brown TM. (2015). Colour as a signal for entraining the mammalian circadian clock. PLoS Biol
13, e1002127.
1386
1387
1388
1389
Watts AG, Swanson LW & Sanchez-Watts G. (1987). Efferent projections of the suprachiasmatic
nucleus: I. Studies using anterograde transport of Phaseolus vulgaris leucoagglutinin in the
rat. J Comp Neurol 258, 204-229.
1390
1391
1392
Wickland C & Turek FW. (1994). Lesions of the thalamic intergeniculate leaflet block activity-induced
phase shifts in the circadian activity rhythm of the golden hamster. Brain Res 660, 293-300.
1393
1394
1395
Wong KY. (2012). A retinal ganglion cell that can signal irradiance continuously for 10 hours. J
Neurosci 32, 11478-11485.
1396
1397
1398
Yamazaki S, Kerbeshian MC, Hocker CG, Block GD & Menaker M. (1998). Rhythmic properties of the
hamster suprachiasmatic nucleus in vivo. J Neurosci 18, 10709-10723.
1399
1400
1401
1402
Yannielli PC, Brewer JM & Harrington ME. (2004). Blockade of the NPY Y5 receptor potentiates
circadian responses to light: complementary in vivo and in vitro studies. Eur J Neurosci 19,
891-897.
1403
1404
1405
Yannielli PC & Harrington ME. (2000). Neuropeptide Y applied in vitro can block the phase shifts
induced by light in vivo. Neuroreport 11, 1587-1591.
1406
1407
1408
Zhang DX & Rusak B. (1989). Photic sensitivity of geniculate neurons that project to the
suprachiasmatic nuclei or the contralateral geniculate. Brain Res 504, 161-164.
1409
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1411
1412
Zhao C, Eisinger B & Gammie SC. (2013). Characterization of GABAergic neurons in the mouse lateral
septum: a double fluorescence in situ hybridization and immunohistochemical study using
tyramide signal amplification. PLoS One 8, e73750.
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Figure Legends
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Figure 1. Modelling the non-image forming visual projection. (A) Central targets of M1-type ipRGCs
visualised via X-gal stained sections from Opn4+/tau-LacZ mice. (B) Sagittal reconstruction of forebrain
M1-type ipRGC axon/terminal labelling intensity, indicating chosen slice angle and thickness: 30° off
the coronal plane, 600 µm thickness. (C) Projected ‘coronal’ view of slice depicted in B flattened
across z-axis. Abbreviations: pretectal olivary nucleus, PON; intergeniculate leaflet/ventral lateral
geniculate nucleus, IGL/vLGN; suprachiasmatic nucleus, SCN.
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Figure 2. Angled slice preparations retain optic tract connectivity across all major non-image
forming nuclei. (A) Image of the recorded side (i.e. side placed facing recording sites) of an acute
angled slice prepared from an Opn4+/tau-lacZ reporter mouse before and (B) after X-gal staining. (C)
Schematic of the slice preparation showing stimulating electrode and pMEA placements used. (D-F)
Electrical stimulation of the optic chiasm (OC) produces widespread excitatory responses across the
pretectum (D), LGN (E) and SCN/peri-SCN (F). Traces represent probability of spike occurrence (from
600 trials) relative to stimulation onset (arrows) at each site on the 59-electrode recording array for
an illustrative slice. (G-I) Bath application of ionotropic glutamate receptor blockers CNQX (20 µM)
and D-AP5 (50 µM) completely abolished the evoked excitatory responses in the pretectum (G; n=21
sites responding to OC stimulation from 3 slices), LGN (H; n=45 sites from 6 slices) and SCN (I; n=68
sites from 2 slices). Left-hand panels show normalised responses to OC stimulation before ( ) and
after ( ) drug application (see methods). Shaded region indicates timepoints where the post-drug
response is significantly reduced (***; P<0.001) relative to baseline (two-way RM ANOVA with
Sidak’s multiple comparison test). Right-hand panels display perievent rasters of multiunit activity
from representative recording sites (green shading = 6 min drug application).
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Figure 3. Angled slices remain viable for more than 24 h and display circadian rhythms in electrical
activity. (A) Image of a representative angled slice preparation illustrating the distribution of pMEA
recording sites within and surrounding the SCN. Red circles indicate sites categorised as being
located in the SCN, blue circles indicate sites categorised as located in the vSPZ. (B) Multiunit firing
rate profiles recorded across the 59 sites of the electrode array from slice in A. Data were binned
(60s) and smoothed with a 2h boxcar filter. (C) Bar chart illustrating the percentage of channels
within the SCN and vSPZ considered rhythmic (based on data from 5 slices; see methods for details
of analysis). Difference in the proportions of rhythmic channels here was analysed by Fisher’s exact
test. (D) Rayleigh vector plots displaying the timing of peak SCN firing activity detected at rhythmic
channels in the SCN (n=100) and vSPZ (n=61). Arrows indicate the median phase and strength of
clustering (r value). Differences in phase and clustering strength were analysed by Mann-Whitney Utest and Browne-Forsythe’s test respectively (analysis performed on linearised data; see methods).
(E) Proportion of channels across the SCN and vSPZ from the above experiments exhibiting
significant changes in firing in response to a 5 min application of 20μM NMDA, applied >26h after
slice preparation. (F) Mean±SEM firing rate across all NMDA-activated channels (n=180), illustrating
robust changes in firing. Grey shaded areas in B and D correspond to the projected timing of the
light/dark transition. ** and *** = P<0.01 and P<0.001 respectively.
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Figure 4. Electrical stimulation of the geniculohypothalamic tract evokes inhibitory and excitatory
responses in the SCN. (A) Schematic of the slice preparation showing stimulation electrode and
pMEA placement used. (B) Electrical stimulation (concentric electrode; 1.2 mA, 200 μs) of the IGL
region evoked inhibitory responses at a subset of SCN recording sites (n=21 sites from 12 slices;
representative recording site shown). (C) Electrical stimulation of the geniculohypothalamic tract
(GHT) also evoked excitatory responses in a second group of SCN recording sites from these 12 slices
(n=27 sites; left panel, representative recording site shown) which could be abolished by bath
application of ionotropic glutamate receptor blockers (CNQX, 20 µM and D-AP5, 50 µM; right panel,
representative recording site shown of 4 SCN recording sites tested). (D) GHT-evoked inhibitory
responses (time to peak, 10 ms moving window) were typically slower and exhibited more variable
timing than excitatory responses. (E) Where tested (n=12 sites), short trains of pulses (5 stimuli at 50
ms, i.e. 20 Hz, or 5 stimuli at 25 ms, 40 Hz) did not evoked significantly larger inhibitions than single
pulse stimulation (one-way ANOVA; P=0.98). (F) GHT-evoked (1.2 mA, 200 µs; 5 x 40 Hz train)
inhibitory responses (middle and bottom panels) but not excitatory responses (top panels) were
blocked by bath application of the GABAAR antagonist, bicuculline (BIC 20 μM; representative SCN
sites shown from a total of 23 excitatory and 5 inhibitory responses tested). (G,H) Quantification of
the effect of BIC and CNQX/D-AP5 relative to predrug values (black bars) for excitatory (G) and
inhibitory responses (H). Data were analysed by paired t-test (except CNQX in G, where only 4 cells
were tested). **=P<0.01; n.s.=P>0.05.
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Figure 5. Optogenetics-based selective activation of geniculohypothalamic signalling. (A) GFPimmunostaining enhanced ChR2/EYFP expression in the thalamus of GAD2-Cre; Ai32 mice, revealed
robust expression in neural processes across the predominantly GABAergic IGL/vLGN region. (B)
Central region of A showing EYFP signal across the dLGN and IGL/vLGN at higher magnification. (C)
Live image of slice showing optical fibre positioning over IGL/LGN (left) and schematic of slice
preparation with optical fibre and pMEA placements used (right). (D, E) Direct optical stimulation
(465 nm LED; ~800mW/mm2 at fibre tip, 10 ms flash – represented by blue shaded area in traces)
evoked robust and rapid excitatory responses across the IGL/vLGN and much weaker responses in
the dLGN. Traces in D represent probability of spike occurrence (from 200 trials) relative to
stimulation onset at each site on the 59-electrode recording array for the slice shown in C. Data in
Ei,ii and iii show perievent rasters and larger spike probability histograms for the traces indicated in
D. (F) Schematic of slice preparation showing optical fibre and pMEA placements used for
investigation of GHT signalling. (G) Optical stimulation of GABAergic GHT projection neurons evoked
modest inhibitory responses in a small subset of SCN recording sites (n=12 sites from 4 slices;
inhibition, mean±SEM: 43.8±5.5% of pre-stimulation; latency, median±SD: 60±25 ms: representative
SCN recording sites are shown in G and H). (H,I) Where tested (n=6), optogenetic-evoked inhibitory
responses were abolished by bath application of the GABAAR antagonist, (+)-bicuculline (20 μM). (H)
data from a representative SCN recording site pre- and post-BIC (upper and lower panels
respectively). (I) mean±SEM response magnitude pre- and post-BIC (n=6). Data were analysed by
paired t-test. **=P<0.01.
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Figure 6. Geniculohypothalamic modulation of SCN responses to optic input. (A) Perievent raster of
SCN multiunit responses to electrical stimulation of the OC; synchronous with (left panel), or 250 ms
after (right panel), optogenetic activation of GABAergic GHT projection neurons (see Fig. 5F for
recording setup). (B) Normalised mean (±SEM) OC-driven responses of SCN subpopulations as a
function of GHT pre-stimulation latency: at most recording sites (n=45/71 OC-responsive sites from 3
slices; see methods for classification details), GHT pre-stimulation evoked long-lasting suppression of
OC-driven responses (two-way RM ANOVA: GHT pre-stimulation, Group and interaction all P<0.001;
asterisks indicate significance differences between groups in Sidak’s post-tests). (C) GHT-driven
inhibition of OC-evoked responses was significantly greater ipsilateral to the optogenetically
stimulated LGN (n=22 vs. n=23 contralateral sites; t-test, P<0.05). (D) Perievent histograms of SCN
responses (perievent rasters are depicted in A) showing enhanced OC-driven responses during the
late day/early projected evening. (E) Normalised mean (±SEM) response of GHT-responsive sites
(n=45) to OC-stimulation: synchronous with ( ), or subsequent to ( ), optical activation of
GABAergic GHT projection neurons. In both cases F-test indicated a significantly better fit to a
sinusoidal (24h) vs. linear function (Both P<0.001 that a linear process generated the data). (F) GHTdriven inhibition of OC-evoked responses (data from E, expressed as a percentage of the response
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observed under synchronous stimulation) varies significantly across the 24 h in vitro recording
period (one-way RM ANOVA of 6 h binned data; P<0.001; asterisks indicate significance differences
between timepoints in Tukey’s post-hoc tests). (G) Perievent raster of IGL/vLGN multiunit responses
to OC and optical stimulation (as in Fig. 5C). (H) Normalised mean (±SEM) IGL/vLGN population
activity (n=34 responding sites from 3 slices) under basal conditions (top panel), following direct
ChR2 (middle panel) or OC stimulation (bottom panel). In none of the above cases did we observe
evidence of pronounced day-night variation (F-test: all P=0.999 that were more likely to have been
generated by a linear vs. sinusoidal model). Shaded areas in A and E-H represent projected night. For
B and F: *, ** and *** represent P<0.05, P<0.01 and P<0.001 respectively.
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Figure 7. Geniculohypothalamic-driven inhibition of SCN responses to optic input involves GABAA
and GABAB receptors. (A) Perievent histograms of representative SCN multiunit responses to
electrical stimulation of the OC at varying latencies of GABAergic GHT neuron pre-stimulation; under
control conditions (top panel), following bath application of the GABAAR antagonist bicuculline (20
µm, BIC; middle panel), or co-application of BIC (20 µM) & the GABABR antagonist CGP55845 (3 µM,
CGP; bottom panel). (B) Normalised mean (±SEM) population responses to OC-stimulation as a
function of GHT pre-stimulation latency (n=44 GHT-responsive sites from a total of 81 OC-responsive
sites in 3 slices). Bath application of BIC substantially attenuated GHT-driven inhibition at short
latencies; residual responses under BIC were further attenuated when co-applied with CGP. (C)
Normalised mean (±SEM) population responses to OC-stimulation in a separate, second experiment
investigating the effects of shorter GHT pre-stimulation latencies (n=14 GHT responsive/21 OCresponsive sites from 3 slices): note that the initial components of the GHT-evoked inhibition were
almost completely attenuated by BIC application alone. Data in B and C were analysed by two-way
RM ANOVA with Sidak’s post-hoc tests (P<0.01 for GHT pre-stimulation, treatment and interaction in
both cases). Black symbols in B and C represent significant differences relative to 0ms delay
(***=P<0.001), Red symbols represent significant differences between BIC and baseline, blue
symbols (in B) represent differences between BIC and BIC+CGP (#=P<0.01, $=P<0.05).
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Figure 8. Geniculohypothalamic signalling modulates specific features of the SCN light response in
vivo. (A) Histological reconstruction of a DiI labelled recording electrode/drug cannula placement in
the LGN (light microscopic image, pseudo-coloured green) and (B) schematic showing recording
electrode/drug cannula placement in LGN and projected spread of drug infusion. (C) Multiunit
responses detected at level of probe in A-B to 5 s light step (405 nm, 15.3 log photons/cm2/s)
presented to both eyes before and after infusion of the GABAA receptor agonist muscimol (1 mM; 23 μl injection at 1 μl/min) completely abolished spiking activity at recording sites up to 750 μm from
cannula tip. (D) Normalised mean (±SEM) SCN population responses to 5 s light steps (405 nm, 15.3
log photons/cm2/s) presented to the contralateral (left eye), ipsilateral (right eye) or both eyes,
before and after infusion of muscimol into the ipsilateral LGN (n=63 sites from 5 animals); schematic
(left panel) shows recording electrode placement in the SCN and projected spread of drug infusion in
the LGN. (E-F) Data as in D for responses following infusion of muscimol into the contralateral LGN
(E; n=33 sites from 3 mice) or under control conditions (F; identical protocol with no drug delivery,
n=127 sites from 6 mice); schematics of the recording set-up are shown on the left. (G-I) Normalised
mean (±SEM) SCN population responses to 5 s light steps of varying intensity presented to the
contralateral (left), ipsilateral (right) or both eyes (left, middle and right panels respectively), before (
) and after muscimol (or no drug) infusion ( ) into the ipsilateral LGN (G), contralateral LGN (H) or
under control conditions (I). For each panel in G-I an F-test was performed to determine whether a
single sigmoidal function could explain the irradiance response relationship pre-and post-muscimol.
This was the case (P>0.05) in all but panel G contralateral and binocular (P=0.001 and 0.002
respectively).
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Figure 9. Optogenetic activation of the geniculohypothalamic tract inhibits specific features of the
SCN light response in vivo. (A; upper pannel) Histological reconstruction of a DiI labelled optrode
placement in the LGN of a GAD2-Cre; Ai32 mouse (DiI in red, ChR2-EYFP in green, DAPI in blue) and
(lower panel) schematic showing optrode placement in LGN and projected spread of optical
illumination (based on 0.66 numerical aperture fibre). (B) Representative perievent histograms
showing responses from optrode in A (responses to 100 trials, for clarity responses shown only for
central 12 sites) to 10ms light flashes (465 nm LED; ~630mW/mm2 at fibre tip, represented by blue
shading). (C) perievent raster for optogenetic responses identified at the site in B indicated by *. (D)
Schematic of recording setup for data in E-G. (E) Normalised mean (±SEM) SCN population responses
to 5 s light steps (405 nm, 15.3 log photons/cm2/s) presented to the contralateral, ipsilateral or both
eyes, in the absence (red) and presence (blue) of optogenetic stimulation of the GHT (for clarity data
for 10Hz trains only are shown). Data are derived from 72 light responsive channels (from 6 mice).
Left panels show initial components of the response (10ms bin size, smoothed with a 50ms boxcar
filter), right panels show full light response (200ms bin size). (F, G) Normalised mean (±SEM) change
in light-evoked SCN activity as a function of GHT stimulation frequency for stimulation of
contralateral, ipsilateral or both eyes, either during early (F, 0-250ms) or later (G, 3-5s after start of
visual stimulation) components of the response. Data were analysed by two-way RM ANOVA with
Sidak’s post-test. For F, ANOVA revealed a significant effect of stimulation frequency (P<0.001) but
not eye or interaction, post-tests indicated all frequencies were significantly reduced compared to
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no GHT stimulation. For G, ANOVA revealed a significant interaction between eye and stimulation
frequency, coloured asterisks represent significant differences relative to the no-GHT stimulation
group (0 Hz) for that eye. (H) Mean (±SEM) light-evoked IGL/vLGN activity from experiments above.
Data are based on changes across full 5s stimulation (from 41 light and optically responsive sites
from 6 mice), analysis performed on the early and later epochs (as in F and G; not shown) produced
qualitatively similar results: two-way RM ANOVA revealed significant interaction (eye x stimulation
frequency; P<0.001). Coloured asterisks represent significant differences relative to the no-GHT
stimulation group for that eye (Sidak’s post-test). (I) Normalised mean (±SEM) IGL/vLGN responses
from dataset above for contralateral and ipsilateral visual stimuli in the absence (red) or presence
(blue) of optogenetic stimulation (for clarity data for 10Hz trains only are shown). Grey shaded areas
in E and I represent darkness. In G and H, ** and *** represent =P<0.01 and P<0.001 respectively.
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Figure 10. Schematic of proposed geniculohypothalamic circuitry and its impact on SCN output. (A)
Based on the data presented in this manuscript, we propose that GABAergic neurons contributing to
the GHT are primarily excited by contralateral retina inputs and convey the resulting inhibitory
signals to SCN neurons that receive input from either (or both) eyes. An SCN-intrinsic mechanism
then adjusts SCN responses to retinal and GHT inputs according to time of day. (B,C) Schematic
illustrating inferred excitatory and inhibitory synaptic influences on SCN neurons (B) and the
resulting spike output under varying illumination (C; upper and lower panels reflect SCN cells
receiving input from just the contra- or ipsilateral eye respectively). Following experimental
illumination of just the contralateral retina (left panels), GHT input is activated and inhibitory inputs
reach the SCN 50-100ms after arrival of excitatory retinal inputs. For SCN cells that receive
contralateral retinal inputs, the resulting long-lasting inhibitory influence attenuates the excitatory
retinal drive leading to a suppression of steady state light-driven firing rates (especially during the
projected day when SCN neurons are more sensitive to GHT signals). By contrast, in the absence of
any driving excitatory input (SCN cells receiving only ipsilateral input), spontaneous firing is
unaffected. When only the ipsilateral retina is illuminated, activity of the GHT is very low and
therefore doesn’t influence SCN responses (middle panels). Under the more commonly encountered
situation in the natural world where both eyes are illuminated (right panels) inhibitory GHT signals
provided by the GHT are able to reduce SCN responses regardless of which eye a cell receives input
from.