BIOL RHYTHMS

Peripheral oscillators

Multiple: Liver, kidney, heart, spleen, muscle capable of producing circadian rhythms & contain clock genes Potentially entrained by main oscillator since can be out of sync. Clock genes in periphery dampen in vitro over time (compared to SCN where rhythms persist)

Outputs of clock: HPA axis

Adrenal gland: Triangular structure on kidneys a) Cortex: Outside, produces: - glucocorticoids: Cortisol stress, glucose release - mineralcorticoids: water intake - sex hormones: spects of puberty like hair Regulated by hormones (slow stress response) b) Medulla: Inside, produces adrenaline, NE. Regulated by sympathetic NS (very quick response) 1) Hypothalamus: Corticotropin releasing factor (CRH) in PVN [input from SCN via SPZ] 2) Anterior pituitary: Adrenocorticotropic hormone (ACTH) through blood 3) Adrenal cortex: Cortisol SCN: Responsible for rhythm of glucocorticoid secretion. Rhythms fluctuate in opposition to melatonin (low at night, high in day), important for rise/food, very different in nocturnal/diurnal STRESS RESPONSES: - Fast (neuronal): adrenal medulla controlled directly by sympathetic NS through direct nerve fibers & adrenaline - Slow (hormonal): HPA axis & cortisol POSSIBLE PATHWAYS: - Direct SCN-PVN connections [through subparaventricular zone] - Indirect SCN-DMH (dorsal medial hypothalamus)-PVN EVIDENCE OF SCN CONTROLLING CORTISOL RHYTHM: - Electrophysiological and anatomical studies - SCN fibers connect to stress-induced c-Fos cells in the PVN - VP into DMH increased corticosterone - Lesion of VP in the SCN increases plasma ACTH and CRH mRNA

Circadian rhythms terms and concepts

Circadian rhythm (close to day): Temp, cortisol, glucose, insulin secretions, stress response etc Entrainment: Synchronization of internal to external environment (adjust frequency/phase to earth's rotational cycle) Ultradian: < 24 hrs (heart rate, glucose/insulin) Infradian: > 24 hrs (lunar) Actogram: Data collector for biological rhythms; pictorial representation of behavior/physiology that appears to follow 24 hr pattern. Need large amount of data across many cycles to determine (ex: wheel running linked to computer # of events and when start, depends on what you're looking at) Freerun: Emergence of internal cycle not controlled by external signal (if longer than 24 hrs, starts a little later every day. Typical) Period (tau): Time units that cycle takes to complete Frequency: # of cycles in a particular time unit (inverse of period) Amplitude: From lowest/highest point range (can diminish or dampen over time if no light changes) Phase: Calculated in relation to other variable, fixed onset of light to onset of behavior

Afferent connections of SCN

Coming into SCN 1) LIGHT INFO: - Retinohypothalamic tract (RHT): SCN receives direct fibers. Directly from temporal retina > ganglion cell axons > through LGN (nucleus of thalamus) > occipital cortex - Geniculo-hypothalamic tract: LGN neurons (from intergeniculate leaflet) go back and innervate the SCN and peri-SCN areas. a) Retinal photoreceptors: rods/cones mediate visual but some blind people can still entrain to L/D, mice with rods and/or cones removed can still entrain, if cut optic nerve cannot (so info coming from retina) b) Melanopsin-expressing ganglion cells: In internal layer of retina (not photoreceptors). Mediates entrainment but rods/cones involved as well - Melanopsin: responds to light but not image-forming info, found in skin of reptiles 2) NON-VISUAL: Many projections from diff areas, most abundant are related to sleep/wake - Median raphe nucleus (same pattern as RHT and GHT) - Paraventricular thalamic nucleus (PVN) - Lateral septum - Ventral medial hypothalamus (VMH)

Efferent connections of SCN

Coming out of SCN Study with anterograde tracing and following peptide routes from SCN 1) DENSEST PROJECTIONS: Localized areas - Peri-SCN - Subparaventricular region - Some connections terminate in dorsal medial hypothalmus (DML) close to ventral medial hypothalamus (VML), posterior hypothalamic area, and periaqueductal gray MBH/DMH (food) 2) FIVE OTHER PATHWAYS: - Thalamic paraventricular - Anterior route to pre-optic area - Septal nuclei - Arcuate and ventromedial hypothalamus (VMH) - IGL and ventral geniculate Connections local to hypothalamus/thalamus, doesn't send direct connections to specific areas

Outputs of clock: Reproductive Cycles

HPG axis: Hypothalamus-pituitary-gonadal axis Rats: Estrus cycle 4-5 days, expression of circadian function. Each afternoon, spike in luteinizing hormone occurs, rupture of follicle and ovulation Same hormones cause release of gonadal in m/f: 1) Hypothalamus: GnRH 2) Anterior pituitary: LH & FSH cause maturation of reproductive cells in m/f 3) Gonads: testosterone (male testes) and estrogen and progesterone (female ovaries) EVIDENCE: - Ovulation can be delayed by 24 hrs if inject anesthetic during critical time window of ovulation - Ovarectomized females have daily surges of LH which is normally obscured by fluctuations of other hormones - LH surge about 4 hrs before activity, if shift circadian rhythm, LH surge shifted by same degree - "Split-rhythm" hamsters have two LH surges before each activity - SCN-lesioned animals lose estrus cycle and don't have LH surges when ovaries removed even if given estrogen MOLECULAR: - Clock + Bmal > ebox and vasopressin > contacts GnRH activate/deactivate > LH - Feedback loop: ebox and vasopressin > kisspeptin > V1a receptor, progesterone receptor, glutamate, GABA (from ovarian signal). SCN sending to GnRH system to produce rhythms CONNECTIVITY: - SCN > anteroventral periventricular nucleus (MPA) to GnRH cells - VIP to GnRH cells - New evidence AVP to kisspeptin

Outputs of clock: Temperature

Higher in daytime, lower at nighttime not dependent on activity - Late development of temp rhythms in rodents - SCN lesion does NOT abolish temp rhythm - Control in anterior hypothalamus - Control by SCN appears to be mediated by projections to the medial pre-optic area (MPOA) - SCN to PVN (melatonin and corticosteriod) sPVz > DMH > LH > wakefulness and feeding

Light as synchronizer

Light not used only to see: - Retinohypothalamic tract (RHT) terminates in SCN (see movement of dyes from photoreceptors) Retinal info goes to thalamus & LGN to occipital lobe for vision, and other areas for movement dilation/contraction - Optic chiasm: axons from retina, cross each other (other areas of hypothalamus also recieve direct lght input)

Biological Clock

Linked to 24 hr cycle but very diverse, altered by light, food, sleep schedules, seems temp compensated (drosophila enclosion not affected) Adapt to env, predict/prepare for changes, internal coherence/organization Found in plants (phaseoleus leaf movements) fruit flies, horse shoe crabs different sensitivty to light, bees location Chemical clocks driven by negative/positive feedback loops (like homeostasis) External env zeitgeber > internal self-sustained oscillator > output rhythms Homeostasis: central to rhythms, fluctuate around set point but can't explain rhythms on it's own because do more than respond to env (differences in hunger patterns, sleep etc)

Studies with SCN

Main evidence of internal oscillator from studies where animals were isolated without any external cues (unchanging env), had internal rhythms of behavior (Richter 1940s first attempt monitored rat behavior) a) Lesions in front part of hypothalamus eliminated rhythmic behavior: bilateral SCN lesions abolish - Suprachiasmatic nucleus: Master oscillator in mammals - Hypothalamus: Small area of brain below thalamus connected to pituitary gland (blood vessels and axons) that controls endocrine glands, metabolism (feeding/drinking) reproduction (gonadal hormones) - Stephan (1972): Lesions in SCN of rats, abolished estrus cycles and behavioral rhythms (small area in anterior hypothalamus 20,000 cells) Lesion with electrolytic lesion: pass strong current through electrode, destroy tissue become arrhythmic - Antelope ground squirrels with SCN lesions were more likely to be killed by preds b) Isolate tissue to see endogenous rhythm c) Rhythm restored when SCN transplanted

Molecular clock Mechanisms

Most important in mammalian SCN: Per, Clock, Bmal, Cry, Tim (at least 10 more) 1) Per gene and protein activation: 3 diff forms of Per, follow 24 hr pattern of expression that changes after organism is exposed to pulse of light capable of producing phase shift 2) Tim/Per concentration increases over wakefulness and decreases towards sleep MAMMALIAN CIRCADIAN CLOCKWORK MODEL: - Has positive/negative feedback loops 1) Clock and Bmal1 form heterodimers (associated proteins) that activate transcription of Per and Cry genes through E-box enhancers (DNA response elements in nucleus) 2) As Per levels increase, form complex with Cry in cytoplasm and are phosphorylated 3) Per-Cry heterodimers migrate to nucleus & associate with Clock-Bmal1 heterodimers and shut them off 4) Clock-Bmal positive activators MUTANT CLOCK GENES: changes in rhythms; shorter or longer endogenous period, arrhythmicity, inability to entrain to a zeitgeber EXPERIMENTS: more than circadian rhythm affected - Mice w/clock mutations sleep 1-2 hrs less - BMAL variants associated with SAD and metabolic disorders - Clock polymorphism associated with "morningness" and hyperactivity - NPAS2: related to timing of sleep - Per 1/3 polymorphism associated with extreme diurnal preference - Per 2 & CK1: Very clearly associated with familial advanced sleep phase [has to sleep much earlier than most, among family] Many of these same genes associated with propensity to cancer, bipolar disorder, metabolic syndrome etc

Functional Organization of SCN

Neurons vs network: SCN neurons considered independent oscillators EVIDENCE: - Same model used in invertebrates - Circadian function initiated before synaptogenesis connects cells [rhythms present even before cells communicate] - Individual SCN neurons maintained in culture will produce certain circadian rhythms of firing MODELS OF SCN FUNCTION: - Light input via RHT > core > shell > output Each cell could be oscillator that light synchronizes OR some cells oscillators in core along with gate cells to pass on info into shell (uncertain). Need mass & both parts to produce rhythms MODELS BASED ON: - Temporal activation of various clock genes during 24 hr cycle and following pulse of light - Pattern of clock genes in the "split rhythm" hamster: "Splitting" can appear over time, and not every animal shows effect. Animals placed in LL initially have >24 hr cycles of activity but then split into two 12 hr bouts. Look at sacrificed brain, differential expression of clock genes btwn SCN sides (each works as independent oscillator, de-synchronized) ANIMATION: Fluorescent green protein (GFP) fused to gene and expressed when gene is expressed (like Per1 circadian pattern large 24 hr fluctuations in core)

Outputs of clock: Pineal Gland

Not brain tissue but located in cranium: From dorsal diencephalic anlage (thalamus/hypothalamus), connected by pineal stalk to diencephalon - Photoreceptive in birds/reptiles, not mammals - Oscillator in birds: if remove, becomes arrhythmic - In all species, pineal innervated by sympathetic NS from superior cervical ganglion PATHWAY: SCN controls in very indirect path, light controls melatonin production SCN > subparaventricular zone > paraventricular nucleus (PVN) > Intermediolateral cell column in spinal cord > superior cervical ganglion (part of sympathetic) > Pineal gland PVN: control sympathetic NS & produces CRH, MELATONIN: Produced in pineal from serotonin/tryptophan [serotonin high during day low at night, inverse with melatonin & enzymes], released in same circadian pattern for nocturnal and diurnal - Feedback system to SCN, has receptors for melatonin [pineal rhythm > melatonin > SCN] - SCN has high neural activity in day, inhibits PVN and subsequently sympathetic NS, - SCN low neural activity in night, allows PVN and sympathetic activation. Releases NE from SCG, activates alpha/beta receptors in pineal to stimulate production of melatonin by controlling the enzyme serotonin N-acetyl-transferase levels - If light activates SCN at night, inhibition of sympathetic occurs, producing inhibition of melatonin in pineal

Characteristics of SCN

OSCILLATOR: Needs 1) Light input: RHT 2) Self-sustained oscillations regardless of connections to other areas of brain in vitro - In vivo: SCN island surgically disconnected from other neural structures continues to oscillate - In vitro: SCN removed entirely and placed in petri dish will continue to have patterns of firing (diff depending on when removed it) a) Rhythms of: - Metabolism/energy use: Measure using 2-deoxy-D-glucose (2-DG) (cell takes up but can't use). Fluctuate with 24 hr rhythm, higher glucose utilization even in nocturnal - Neuronal firing: spontaneous rhythms of firing in many rodents (high in day low at night) even in nocturnal or placed in LL or DD therefore intrinsic b) Contains genes/proteins that are the clock mechanisms: Per, CRY, Tim, Clock, Bmal (Drosophila) c) Restore rhythms by fetal SCN transplantation - Consider location (close to original), type of tissue (SCN), dispersed or encapsulated (restores but not as well) - Period of restored rhythm matches donor, not host ex: tau mutant hamsters long/short rhythms, graft sets rhythm (remove tissue, place in needle, inject into host). - Recovery rhythm not as precise/dampened - New SCN not innervated by RHT so not synchronized

Chronobiology: History

Part of biological science, study of internal clocks and external rhythms in organisms Clock: Undergoes regular cycles of motion, has to be precise and temp compensated (non-linear) HISTORY: Clocks/calendars significant, mostly based around planetary movements (day/night) ex: obelisks, hemicycle, star clocks, water thief clocks, labyrinth, lunations (calculate dates). Synchronize society MECHANICAL CLOCKS: diversity of mechanisms (mercury drops, drum cables, falling weights with chimes) Galelio isochronism of swinging pendulum later replaced by coiled spring Has to be able to keep steady oscillation (temp changes it, needs to be temp compensated for seasonal changes) and sea voyages John Harrison construct chronometer, time gives location/space in time/direction MODERN CLOCKS: balance wheel, electric pulses, vibration of quartz Atomic clock: Resonance rate of hydrogen/rubidium/cesium, not perfect but used by GPS considered universal coordinator of time (still need leap seconds though)

Entrainment

Phase control and period adjustment REQUIREMENTS: - Sensitivity to some (but not all) env stimuli - Presence of zeitgeber - Experimental demonstration of entrainment; constant relationship btwn period of endogenous rhythm (tau) and period of zeitgeber (T), no masking In nature, achieve entrainment by sampling light levels at dusk/dawn (look out) Phase response curve (PRC): Quantify entrainment. Graphically represents phase shifts in response to single pulses of light given at certain phases of the intrinsic rhythm. Generally similar across species (depends on intensity, duration, wavelength) Flat, dip down, dip up. - CT0: Arbitrary, phase in cycle under entrained conditions, refers to lights on - Time on X, size of phase shifts on Y Give light pulse in DD: - In subjective day: no shift - At early subjective night: Phase delay (like saying night hasn't started yet, wait) - At late subjective night: Phase advance (like saying night is over already, be earlier next time) Indicates sensitive periods when light makes us more likely to shift rhythms NON-PHOTIC ENTRAINMENT: - very different from photic PRC, chemicals can cause phase shifts (like exercise arousal, drugs) may be mediated by serotonin/GHT. Produces phase delay when exposed in day but not night (therefore probably different mechanisms) Masking: Appears to be entrainment, but as soon as external cue removed, restores phase shift as if hadn't been exposed (apparent synchronization, not true)

Molecular response of phase shifts

Production of Fos in core & Per activation Fos: Protein produced when the neuron is activated (general response. Only happens if light pulse capable of producing phase shift)

Neurotransmitters in the RHT

REQUIREMENTS: - Present in retinal ganglion cells - Released during light stimulation - Produce similar effects to light on SCN (phase shifts, changes in spontaneous firing rate, initiate cascade of events similar to light i.e. Fos expression, Per) - Blocked by specific antagonists that block effects CANDIDATES: - Glutamate - Pituitary adenlyate cyclase activation polypeptide (PACAP) EVIDENCE: - Glutamate in retinal ganglion cells; subset co-expresses PACAP - GLU and PACAP distributed throughout retina - GLU receptors in SCN: NMDA, AMPA metabotropic receptors in core and shell - PACAP receptors 1 and 2 in SCN - Released by light stimulation: Electrical stimulation of optic nerve - Endogenous rhythm of GLU: High at night and low at day - Things that mimic GLU produce similar effects (time of day dependent) and if put GLU into SCN: Entrainment via phase shifts, light pulses that induce phase shifts produce Fos & phosphorylation of CREB & Per protein - Glutamate agonist mircoinjected in SCN region produces phase shifts in hamsters (Pathway involves nitric oxide and phosphorylation of CREB protein) - Activation of NMDA agonists induces c-Fos in early and late night - NDMA antagonists reduce c-Fos expression and pCREB induction after a pulse of light - If give GLU NMDA agonist instead of light pulse, produces phase shift and Fos, give antagonist no more phase shift or Fos - NMDA antagonists that block glutamate also block Per induction to light stimulation

SCN anatomy

Small, tightly packed cells in hypothalamus & third ventricle, just above optic chiasm (10 thousand on each side, 2 SCNs). Differ in core/shell areas CHEMOARCHITECTURE: - Core (input): Expresses vasoactive intestinal polypeptide (VIP), NPY, and gastrin-releasing peptide (GRP). Receives retinal input and connects to shell - Shell (output): Expresses vasopresin (VP/aVP). Sends fibers to subparaventricular area (output system just above SCN)