Tracing the ultimate timekeeper: Pathways involving the mammalian suprachiasmatic nucleus

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Tracing the ultimate timekeeper Pathways
involving the mammalian suprachiasmatic nucleus

• Lianne K. Morris-Smith
• NSB275
• 4/26/2005

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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 free-running 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 (sleep-wake 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

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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)
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Big Picture Goal

• To unravel the anatomical and chemical pathways
  by which the SCN exerts its timekeeping influence
  on physiological and behavioral phenomena.

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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)

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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)

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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)

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Conclusion

• Most RGCs that project to SCN are
  melanopsin-positive (Opn4), therefore melanopsin
  is a prime candidate for the photopigment
  mediating photic circadian entrainment.

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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)

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Opn4 RGCs and their retinal distribution
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Opn4 RGCs displaced vs. nondisplaced
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Axonal projections of Opn4 RGCs
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Intrinsic photosensitivity of Opn4 RGCs
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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

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II. Retinal output Distribution of Opn4 RGCs
in their various hypothalamic targets

• A broad role for melanopsin in nonvisual
• photoreception (Gooley et al., 2003)

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• 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

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Anterograde tracing from retina
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Novel retinal projections to the vSPZ and the VLPO
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Percentage colocalization of melanopsin-positive
and retrogradely-labeled RGCs
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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 RGCs also project to vSPZ and VLPO

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Roles for melanopsin-positive retinohypothalamic
projections
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III. Hypothalamic outputDistribution 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)

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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?

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• Methods
• Fluorescent immunohistochemistry
• Biotinylated dextran (BD) anterograde tracer
• CTB or FG retrograde tracer
• In situ hybridization
• Anti-TRH (DMH neuron marker), anti-orexin-A,
  anti-glutamate, anti-GAD67 (GABA synthase marker)
• Excitotoxic lesions ibotenic acid
• Activity recordings
• EEGs and EMGs
• Serum hormone measurements
• Cell counting

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Correlation of circadian indices with extent of
DMH lesion
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Effect of DMH lesions on endocrine rhythmicity
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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

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Major DMH pathways involved in circadian timing
of sleep-wakefulness and hormonal secretion
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Conclusions

• DMH has an overall activating (arousing) role
• DMH integrates circadian timing information with
  internal and environmental signals to influence
  animals behavioral state

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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)

• 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

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Examples of retrograde labeling in the SCN and
anterograde labeling in selected centers (MPA,
A-D sPVZ E-H) of the arousal system
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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

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• 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

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Conclusion

• sVPZ, DMH and MPA serve as interface between SCN
  and diverse physiological systems, including the
  sleep-wake system

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Potential indirect SCN output pathways to major
sleep- and arousal-regulatory nuclei
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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)

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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)