Tracing the ultimate timekeeper: Pathways involving the mammalian suprachiasmatic nucleus Lianne K. Morris-Smith NS&B2754/26/2005

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 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

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)

 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%) Summary of Results

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

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 melanopsin- positive 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+ RGCs also project to vSPZ and VLPO

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, anti-glutamate, 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 has an overall activating (arousing) role ► DMH integrates circadian timing information with internal and environmental signals to influence animal’s behavioral state

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

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 arousal- regulatory 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)