Transcript Slide 1

Tracing the ultimate timekeeper:
Pathways involving the mammalian
suprachiasmatic nucleus
Lianne K. Morris-Smith
NS&B275
4/26/2005
Background
Ablating SCN abolishes daily sleep-wake rhythms but does not affect total
amount of sleep or wakefulness: SCN does not maintain behavioral states but
controls their timing (reviewed by Deurveilher and Semba, 2005)
Without photic stimulation or nonphotic zeitgebers, circadian rhythm is freerunning: SCN is an endogenous pacemaker
Transgenic mice that lack rods and cones are functionally blind but are still
able to show photic entrainment (reviewed by Gooley et al, 2003)
Rods and cones not needed for light-induced circadian entrainment (sleepwake cycles, negative masking of locomotor activity, suppression of pineal
melatonin) or the pupillary light reflex (reviewed by Gooley et al, 2003)
Melanopsin, a novel photopigment, found in mammalian inner retina
(reviewed by Gooley et al, 2003)
SCN efferents mainly confined to hypothalamus (reviewed by Deurveilher
and Semba, 2005) yet has widespread influence
Seasonal variation in circadian
rhythm of Finnish bats
Nocturnal during warm
summer months.
Diurnal during colder
spring and fall (fewer
insects on colder nights,
plus less competition and
predation in day)
(grey = night hours
black = active hours
reviewed by Saper et al., 2005)
Big Picture Goal
To unravel the anatomical and chemical
pathways by which the SCN exerts its
timekeeping influence on physiological
and behavioral phenomena.
I. Melanopsin-positive RGCs and the
retinohypothalamic tract
► Melanopsin
in cells of origin of the
retinohypothalamic tract (Gooley et al.,
2001)
► Melanopsin-containing
retinal ganglion
cells: architecture, projections and
intrinsic photosensitivity (Hattar et al.,
2002)
Melanopsin in cells of origin of the
hypothalamic tract (Gooley et al. 2001)

Do RGCs that express melanopsin project to the SCN?

Methods:
 Immunocytochemistry (FluoroGold, FG, a retrograde
tracer in right SCN of rats)
 In situ hybridization (melanopsin riboprobe in
retina)
Summary of Results
Most FG-labeled RGCs express
melanopsin mRNA (74.2 ± 0.3%); similar
amount of double-labeling in both eyes.
Most RGCs that express melanopsin
mRNA were FG-labeled (~ 70%)
Conclusion
 Most RGCs that project to SCN are
melanopsin-positive (Opn4+), therefore
melanopsin is a prime candidate for the
photopigment mediating photic circadian
entrainment.
Melanopsin-containing retinal ganglion cells:
architecture, projections and intrinsic
photosensitivity ( Hattar et al., 2002)
► Where
is melanopsin expressed in Opn4+ RGCs?
► Do double-labeled neurons (Opn4+ RGCs) show intrinsic
light sensitivity?
► Methods:
 Lucifer yellow, an intracellular dye
 Fluorescent labeling
► propidium
iodide, a nuclear stain.
► rat anti-melanopsin
► tau-lacZ, a fusion protein (heterozygous insert in
mouse Opn4 gene locus)
 Calcium blockers (Ames medium, cobalt chloride);
glutamatergic blockers (APB, DNQX, APV)
Opn4+ RGCs and their retinal
distribution
Opn4+ RGCs:
displaced vs. nondisplaced
Axonal projections of Opn4+ RGCs
Intrinsic photosensitivity of
Opn4+ RGCs
Summary of Results
►
Melanopsin expressed in soma, dendrites and proximal
axons of Opn4+ RGCs, mainly on cell membranes
►
Most (~ 95%) “nondisplaced” Opn4+ RGCs found in
ganglion cell layer of retina; small proportion of
“displaced” cells found in inner nuclear layer
►
Highest proportion of Opn4+ RGCs in superior,
temporal retina
►
Opn4+ RGCs project bilaterally to SCN, IGL and OPN.
Sparse labeling seen in VLG.
►
Intrinsically photosensitive cells are invariably Opn+
II. Retinal output:
Distribution of Opn4+ RGCs in their
various hypothalamic targets
 A broad role for melanopsin in nonvisual
photoreception (Gooley et al., 2003)
► Do
Opn4+ RGCs project to other
retinorecipient regions besides the SCN, IGL
and OPN?
► What is the distribution of Opn4+ RGCs in
their various hypothalamic targets?
► Methods:
 Recombinant adeno-associated virus containing green
fluorescent protein reporter gene (rAAV-GFP) – fluorescent
anterograde tracer
 Cholera toxin B (CTB) – fluorescent anterograde and
retrograde tracer
 FluoroGold (FG) – a fluorescent retrograde tracer
 Mouse melanopsin riboprobe – in situ hybridization
 Cell-counting
Anterograde tracing from retina
Novel retinal projections to the
vSPZ and the VLPO
Percentage colocalization of melanopsinpositive and retrogradely-labeled RGCs
Summary of Results
► Most
Opn4+ RGCs project bilaterally to
the SCN and contralaterally to the PTA;
almost 20% project to ipsilateral IGL
►~
20% Opn4+ RGCs send collateral
projections to both SCN and PTA
► Opn4+
VLPO
RGCs also project to vSPZ and
Roles for melanopsin-positive
retinohypothalamic projections
III. Hypothalamic output:
Distribution of efferents from Opn4+
RGC-targeted hypothalamic areas
►
Critical role of dorsomedial hypothalamic
nucleus in a wide range of behavioral circadian
rhythms (Chou et al., 2003)
►
Indirect projections from the suprachiasmatic
nucleus to major arousal-promoting cell
groups in rat: implications for the circadian
control of behavioural state (Deurveilher and
Semba, 2005)
Critical role of dorsomedial hypothalamic
nucleus in a wide range of behavioral
circadian rhythms (Chou et al., 2003)
►
Does the dorsomedial hypothalamus (DMH), which
receives parallel projections from both the SCN and
vSPZ, influence circadian control of sleep-wake
indicators (locomotor activity [LMA], feeding, Tb,
corticosteroid secretion and melatonin secretion)?
►
What are the neurotransmitters involved in DMH
projections to VLPO and LHA?
► Methods
 Fluorescent immunohistochemistry
► Biotinylated dextran (BD) – anterograde tracer
► CTB or FG – retrograde tracer
 In situ hybridization
► Anti-TRH (DMH neuron marker), anti-orexin-A, antiglutamate, anti-GAD67 (GABA synthase marker)
 Excitotoxic lesions – ibotenic acid
 Activity recordings
► EEGs and EMGs
 Serum hormone measurements
 Cell counting
Correlation of circadian indices
with extent of DMH lesion
Effect of DMH lesions on
endocrine rhythmicity
Summary of Results
►
►
►
►
►
►
►
DMH lesions considerably reduce circadian rhythms
of sleep-wake behaviors and LMA
Lesions notably reduce total wakefulness, LMA and
body temperature (Tb)
DMH lesions disrupt feeding cycle with little effect
on intake amount.
Lesions eliminate circadian rhythm of corticosteroid
secretion and reduced average daily cortisol levels
by ~ half.
No significant effect on Tb rhythm
No significant effect on melatonin secretion or
rhythm
DMH primarily sends glutamatergic and TRH neurons
to LHA; sends primarily GABAergic projections to
VLPO
Major DMH pathways involved in
circadian timing of sleep-wakefulness
and hormonal secretion
Conclusions
► DMH
► DMH
has an overall activating (arousing) role
integrates circadian timing information
with internal and environmental signals to
influence animal’s behavioral state
Indirect projections from the
suprachiasmatic nucleus to major arousalpromoting cell groups in rat: implications for
the circadian control of behavioural state
(Deurveilher and Semba, 2005)
►
What are the pathways that relay SCN output to the
major wake-promoting neuronal groups: basal
forebrain and mesopontine cholinergic neurons;
posterior hypothalamus orexin neurons;
tuberomamillary nucleus, VTA, SNpc, dorsal raphe
and locus coeruleus aminergic neurons?
►
Methods:
 Tracers: BDA (anterograde) and CTB (retrograde)
 In situ hybridization
► Antibodies to proteins characteristically expressed by
neurons of interest
Examples of retrograde labeling in the SCN and
anterograde labeling in selected centers
(MPA, A-D; sPVZ E-H) of the arousal system
Summary of Results
►
Medial preoptic area (MPA)
 Receives strong SCN projections
 Provides strong projections to forebrain regions: orexin field,
histaminergic tuberomamillary nucleus; projections to
substantia innominata are primarily from rostral MPA
 Provides strong projections brainstem: locus coeruleus region
 Thus, MPA is a strong relay candidate
►
sPVZ
 Receives dense SCN projections
 Sends dense projections to orexin field and tuberomamillary
nucleus
 No brainstem projections
 sVPZ is strong relay candidate
►
Dorsomedial hypothalamus (DMH)
 Receives dense SCN projections
 Sends dense projections to orexin field and tuberomamillary
nucleus
 Caudal DMH projects to brainstem regions: VTA, dorsal
raphe, laterodorsal tegmental nucleus, locus coeruleus
 DMH is strong relay candidate
►
Posterior hypothalamus (PH)
 Receives limited SCN projections
 Sparse to no projections to areas of interest
Conclusion
► sVPZ,
DMH and MPA serve as interface
between SCN and diverse physiological
systems, including the sleep-wake
system
Potential indirect SCN output
pathways to major sleep- and arousalregulatory nuclei
Why is everything so damn
complicated?!
► Multiple
direct and indirect routes may allow
for amplification of the circadian signal and
integration of SCN timekeeping with external
inputs (Deurveilher and Semba, 2005)
► Bottom
line: complexity allows animals greater
adaptability to internal and environmental
conditions, e.g., ambient temperature and food
availability (Saper et al., 2005)
Papers reviewed
►
►
►
►
►
►
Melanopsin in cells of origin of the retinohypothalamic tract
(Gooley et al., 2001)
Melanopsin-containing retinal ganglion cells: architecture,
projections and intrinsic photosensitivity (Hattar et al., 2002)
A broad role for melanopsin in nonvisual photoreception
(Gooley et al., 2003)
Critical role of dorsomedial hypothalamic nucleus in a wide
range of behavioral circadian rhythms (Chou et al., 2003)
Indirect projections from the suprachiasmatic nucleus to major
arousal-promoting cell groups in rat: implications for the
circadian control of behavioural state (Deurveilher and Semba,
2005)
The hypothalamic integrator for circadian rhythms (Saper et
al., 2005)