Welcome to Bi !

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2 Welcome to Bi ! Professors: Paul Patterson Kai Zinn TAs: Janna Nawroth Yanan Sui

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4 Student presentations Select a topic soon (within <2 weeks) and let the TAs know what your choice is. Papers for student presentations are listed at the end of the lecture notes. If you need a paper in advance of the lecture to be given on your topic the prof giving the lecture and he can choose one for you. Presentations are to be rehearsed and worked out in detail with a TA. How well this is done will determine part of your presentation grade. Since there is limited time for presentations, it is essential to have a polished presentation that will be completed within 15 minutes. There will be a few minutes for questions and discussion after each presentation. The participation grade is determined primarily by the extent to which you participate in discussions after presentations.

5 Quizzes The quizzes are given on most Fridays, and will take about 5 minutes at the beginning of the lecture period. They will be very short (~3 questions), each of which can be answered in a few words. Each quiz will cover material from the previous 3 lectures. Their purpose is to make sure you are attending lectures and keeping up with reading. If you have done this, the quizzes should be very easy.

6 Final Prior to finals week, we will hand out a list of questions, answerable with short essays (1-2 paragraphs). These will be drawn from lectures, required reviews, and student presentation papers. The actual final will consist of a subset of these questions. It will be take-home, and will probably have a 3 hr. time limit. You cannot consult lecture notes, reviews, or papers during the exam. We may allow you to use notes that you prepare yourself on the study questions (have not determined this yet). If you have studied and adequately prepared an answer for each question on the study list, you should get 100% on the final.

7 Syllabus (subject to change) Circadian Rhythms, Eating 1/4 Intro/biological clocks [KZ] 1/6 Eating: leptins, genetics of obesity, anorexia [KZ] Social behavior & aggression 1/9 Olfaction [KZ] 1/11 Reward & addiction [KZ] 1/13 Complex genetic control of animal behavior [KZ] 1/16 MLK holiday 1/18 Aggression, stress and depression [PHP] Sex 1/20 Sexual differentiation of the mammalian brain & behavior [PHP] 1/23 Hormones and birdsong; maternal behavior; monogamy [PHP] 1/25 Student presentations + QUIZ Learning and Memory 1/27 The hippocampus and memory [Thanos Siapas] 1/30 Molecular biology of cognition [Alcino Silva] 2/1 Learning/synaptic plasticity I [KZ] 2/3 Student Presentations + QUIZ

8 2/6 Neurogenesis in the adult brain [PHP] 2/8 Sleep & narcolepsy [David Prober] 2/10 Student Presentations + QUIZ Mental Illness & Neurodegeneration 2/13 Prion diseases [KZ] 2/15 Movement disorders: Parkinson's disease [PHP] 2/17 Student Presentations + QUIZ 2/20 President s Day holiday 2/22 Movement disorders: Huntington's disease [PHP] 2/24 Student Presentations + QUIZ 2/27 Dementia: Alzheimer's disease [PHP] 2/29 Disordered thought: Schizophrenia [PHP] 3/2 Student Presentations + QUIZ 3/5 Fear and emotion [David Anderson] 3/7 Genes & environmental control of human behavior {KZ] 3/9 Student presentations + QUIZ Final exam out; due 3/17 noon

9 Policies on use of resources and on collaboration Course: Instructor: Head TA: This sheet outlines the default course policies for problem sets, extra-credit quizzes, and tests. These may be overridden by other instructions in particular instances. However, by default, you are expected to follow these policies. If you have any questions, ASK! Ignorance and confusion are not excuses. Problem Sets Exams Books While working, you may consult: Required texts Y N Y Recommended texts Y N Y Textbooks from prerequisite classes Y N Y English language dictionary (electronic, hard copy) Y Y Y Reference books (CRC, Merck Index, etc.) Y N Y Any other text (State which reference you used) Y N Y Computer and Internet: You may use a computer as a word processor Y Y Y You may use the Internet Y N Y Notes You may use: Your class notes (taken in lecture) Y N Y Hand copies of the class notes of others Y N Y The class notes of others (original or Xeroxed) Y N Y Anything written in your own hand Y N Y Class handouts Y N Y TA/section handouts Y N Y Homework/exams of past years N N N Homework/exams of this year Y N Y Solutions to homework/exams of past years N N N Computational aids For computational aids, you may use: Calculators, computer as calculator, and slide rules Y Y Y Mathematical reference tables (integrals, Laplace transforms, etc) Y Y Y Collaboration The following types of collaboration are allowed: Tell another student that the question exists Y Y N Basic discussion of the problems Y N N Look at communal materials while writing up solutions Y N N Look at other s individual work (i.e. writeups) N N N Sharing a session on Swiss-PDB, Squid Axon tutorial, or other N N N computer-based exercise Turn in a set with more than one name on it N N N Sharing communications from/to TAs Y N N Comparing answers to completed problems Y N N Additional Comments: For problem sets, (1) You must answer in your own words. (2) In addition to these guidelines and rules, obvious copying (even a single sentence) is not allowed. (3) You must feel that you can personally reconstruct the entire response. Extra-credit quizzes

10 Bi156 lecture 1, 1/4/12 Biological Clocks

11 Overview of the mammalian clock retina (light input) --> central clock (hypothalamus)--> cyclical hormone release, cyclical neuronal firing --> synchronization of peripheral clocks in other cell types --> behavioral outputs (conscious outputs include feeding, sleep, exercise preferences). Many unconscious cellular processes (e.g., blood pressure, digestive enzyme synthesis, etc.) are also under circadian control. Up to 10% of all genes show daily cycling in expression level that is controlled by the clock.

12 Common elements of clocks central free-running clock, with a near-24 hr. periodicity, in a particular set of brain neurons phase-shifting inputs from the periphery (light, temperature (in coldblooded animals and invertebrates), hormones, food, exercise). These are called Zeitgebers. behavioral outputs (clock-controlled genes in central clock region cause rhythmic release of humoral factors, regulating clocks in peripheral tissues, so that outputs of these tissues are under circadian control)

13 Elements of a free-running clock mechanism a biochemical process that has built-in fixed delays does not come to equilibrium, but rather cycles back to its original state after 24 hr. many examples of such processes exist (in simplest form, cycling chemical reactions) all known clocks reside within single cells

14 Identification of genes encoding clock components in animals This was done, initially in Drosophila, by isolating mutations that change the characteristic circadian activity patterns of the animal. Cloning of these genes and characterization of their products defined clock mechanisms.

15 Clock phenotypes in Drosophila Konopka and Benzer (1970) of Caltech searched for mutations that would cause flies to eclose (hatch out) at abnormal times identified period (per), which is part of the central pacemaker 3 types of per alleles: per-0 (arrythmic), pers (short period), per-l (long period). Cyclical activity pattern is seen in actogram at left, in which interruption of an infrared beam by a moving fly is scored as a black line. Flies maintained in 12 hr light/12 hr dark, then shifted to constant dark at red arrow. Wild-type flies continue to cycle with a period of slightly less than 24 hr. per-0 flies immediately become arrhythmic upon shift to darkness, showing that they have no freerunning clock.

16 Drosophila genes encoding essential components of the PER clock. period (per) Mutations alter rhythmicity (arrhythmic, long- and shortperiod alleles). RNA and protein cycle. Physically associates with TIM. timeless (tim) Mutations alter rhythmicity (arrhythmic, long- and shortperiod alleles). RNA and protein cycle. Physically associates with PER. Stabilizes PER. double-time (dbt) Mutations alter rhythmicity. Constitutively expressed. Protein kinase (CK1). Physically associates with PER and PER/TIM complexes. Promotes phosphorylation and degradation of TIM-free PER in the cytoplasm and nucleus.

17 Fundamental clock mechanism per/tim mrnas are translated into proteins which are unstable Per is phosphorylated by Dbt, and Tim by Sgg and other kinases, and the phosphorylated forms are less stable than the dephosphorylated ones. Per is dephosphorylated by PP2A which opposes the action of Dbt. Per and Tim have cytoplasmic localizing determinants (CLDs), keeping them out of nucleus when the accumulation rate of Per and Tim outruns the degradation rate Per and Tim heterodimerize, shielding CLDs Per/Tim heterodimer enters nucleus, where it shuts off per/tim transcription per, tim mrna and then protein levels begin to drop Per/Tim complex in nucleus is degraded, so per, tim promoters switch on again

18 Timing of per/tim cycle

19 How does the Per/Tim complex repress transcription of the per and tim promoters? Per contains a domain called PAS, which has counterparts in transcriptional regulators that have DNA-binding domains. The PAS domain is required for dimerization in these other regulators, and only the dimeric proteins can bind DNA and regulate transcription. However, Per has no DNA-binding domain. This suggested that Per works by heterodimerizing with a PAS-domain containing transcriptional activator, and that the Per-containing heterodimer might be unable to switch on transcription. Per would thus act to repress transcription of genes that would normally be switched on by the PAS domain activator.

20 The bhlh-pas domain activator regulated by Per Clock (Clk) A bhlh (DNA-binding region)-pas domain protein. Mutations alter rhythmicity. RNA and protein cycle. Physically associates with Cyc. Cycle (cyc) A bhlh-pas domain protein. Mutations alter rhythmicity. Constitutively expressed. Physically associates with Clk. Clk/Cyc complex binds to E-boxes, which are bhlh protein-binding elements. Clk/Cyc activates transcription of per and tim genes via E-boxes in their promoters.

21 Regulation of Clk/Cyc activity by Per/Tim The bhlh-pas heterodimer Clock/Cycle activates per, tim transcription via E-box binding. Nuclear Per/Tim (or Per alone) inhibits the function of Clk/Cyc, possibly by direct interaction of the Per PAS domain with Clk and/or Cyc. Thus, nuclear Per/Tim causes transcription of per, tim genes to shut off. In addition, Clk/Cyc represses the Clk promoter, possibly by an indirect mechanism. Per/Tim association with Clk/Cyc also blocks autorepression of Clk promoter by Clk/Cyc. Per/Tim thus negatively regulate their own promoters and positively regulate the Clk promoter. This causes Clk transcription to cycle in antiphase to per/tim transcription.

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23 The second loop of the clock The mechanism described produces a functioning clock. However, in both flies and mammals there is a second transcriptional loop that regulates rhythmic Clk transcription. This may be required to produce robust cycling, especially in the native environment. In flies, the second loop involves two other transcriptional regulators, Vrille and PDP. The vri and pdp genes are both switched on by Clk/Cyc, and Vri and PDP both regulate the Clk promoter.

24 The two-loop Drosophila clock Vri represses Clk transcription, and later PDP activates Clk transcription. This difference in the timing of the effects of Vri and PDP provides an independent mechanism for rhythmic alterations in Clk/Cyc activity.

25 Cycling of Drosophila clock components The activities of the clock proteins produce cycling of the clock components. CLK protein and Clk mrna are produced at about the same time, while PER/TIM protein peaks are delayed relative to mrna peaks due to the instability of these proteins.

26 Mouse and fly actograms Mice have activity rhythms very similar to those in flies. However, activity occurs at night in mice,which are nocturnal. As in flies, the natural period in constant darkness is slightly less than 24 hr.

27 Conservation of the mammalian and fly clock mechanisms Remarkably, the central clock mechanism (first loop) is conserved between mammals and flies. The mammalian clock uses Per, Clock, Cyc, and Dbt orthologs. As in flies, Per is unstable, its stability is regulated by phosphorylation, and it blocks the activity of the Clk/Cyc heterodimer. A mutation in human Per2 that eliminates a phosphorylation site and thus affects the kinetics of degradation causes familial advanced sleepphase syndrome, in which people wake up at 3 AM. Clk/Cyc turns on the per promoter.

28 Differences between mammalian and fly central clocks The logic of the two-loop mammalian clock is similar to that of the fly clock. However, the mammalian clock uses Cryptochrome instead of Tim as the major Per partner. In mammals, the Cyc ortholog (BMAL/MOP3) cycles instead of Clk. The second loop of the mammalian clock uses completely different components (nuclear hormone receptors REV-ERB and ROR).

29 The two-loop mammalian clock

30 Mammalian and fly clocks circuits: summary

31 The central fly clock (pacemaker) Which neurons comprise the central clock that runs the fly s behavioral outputs? Only a few small groups of brain neurons express PER protein. Expression of PER in s-lnv neurons only (or even in one of these neurons) is sufficient to drive rhythm of the entire animal. s-lnv cells are thus the master clock neurons.

32 How does the LNv neuron clock drive behavioral rhythms? LNv neurons make a neuropeptide called PDF. The PDF gene is clockcontrolled, and PDF is rhythmically released by LNv terminals. PDF is required for rhythmicity, because flies lacking PDF or its receptor (PDFR) become arrhythmic. Neurons expressing the PDF receptor must connect to circuits that control behavioral outputs. Some of these PDFR+ neurons are themselves clock cells, and PDF helps to synchronize the rhythms of neurons within the clock circuit.

33 Light during normal dark periods resets the clock Animal clocks are all reset by illumination during normal dark periods. This allows the animal to adjust to changes in light/dark patterns. Normally these changes are small, since sunrise and sunset times change by only a few minutes per day during the progression of the seasons. Failure of the human clock to adjust immediately to large alterations in the light/dark cycle after rapid east-west travel causes jetlag. In both mammals and flies, light resetting does not require photoreceptors, so it must be able to occur via light absorption through a photopigment other than rhodopsin.

34 Resetting of the fly clock by light Light during a normal dark period resets the clock by causing rapid degradation of Tim protein. Since Tim levels are normally lowest during the day and Per stability is Tim-dependent, this shifts the clock so that it now resets to a daytime condition. This can represent either an advance or a delay of the clock depending on whether Tim levels are rising or falling at the time of the light stimulus.

35 Light resetting relative to the cycling of fly clock components. Light results in rapid TIM proteolysis, leading to PER loss. If TIM levels are rising, this results in delay back to the previous day phase. If TIM levels are falling, this causes an advance into the next day phase.

36 Characteristics of clock resetting by light in the fly The fly has cell-autonomous Clock/Cyc 24 hr oscillators in many cell types, and surprisingly these can be reset by light in culture, implying that the relevant resetting photoreceptor is widely expressed. The action spectrum of resetting implies that this photoreceptor is most sensitive to blue light, unlike rhodopsin. Behavior in eyeless flies is much less sensitive to light entrainment, so implication is that pathways through the eye are also involved in normal flies, but are not absolutely required. This suggests that behavioral responses may be controlled in a redundant manner, so that loss of the blue photoreceptor would not cause global arrhythmia.

37 The fly clock photoreceptor A genetic screen that took this redundancy into account identified cryptochrome (Cry). Cry has 2 chromophores that absorb blue light. It is homologous to bacterial photolyases, enzymes that catalyze one-step photorepair of DNA lesions. Cry itself cycles, and cry transcription is controlled by the clock system. Cry binds to Tim in a light-dependent manner, and probably facilitates rapid degradation of Tim in response to a light pulse. In cry mutants Per and Tim still cycle in LNs. LN cycling in cry mutants is due to secondary input from eyes to LNs. Thus, although cry single mutant flies entrain behaviorally to LD cycles, double mutants (norp-a cry) lacking eye phototransduction as well lose rhythmicity.

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39 The mammalian clock: anatomy The mammalian central clock is in the suprachiasmatic nucleus (SCN) of the hypothalamus. Light input into SCN is exclusively through retina via retino-hypothalamic connections. SCN lesions can render the animal arrhythmic. Rhythmic SCN output (neuronal firing and hormone release) regulates all circadian properties of the animal.

40 The hypothalamus regulates endocrine system & autonomic nervous system via hormone secretion contains circuits controlling body temperature, heart rate, blood pressure, blood osmolarity, feeding, drinking, circadian rhythms, sex, emotion, etc. has bidirectional communication with cortex, brainstem, etc. divided into 3 regions, each of which is subdivided into many nuclei. has an intimate connection with the pituitary gland.

41 Release of peptides from the hypothalamus many hypothalamic neurons are peptidergic; these peptides may act as neurotransmitters, thus influencing distant areas of the brain peptides can also be released into general circulation as hormones. can be released directly into bloodstream from hypothalamic terminals in the posterior pituitary gland (e.g., oxytocin, vasopressin) can also be secreted into vessels of the anterior pituitary, causing release or inhibition of release of secondary pituitary hormones which enter the bloodstream (e.g., luteinizing hormone releasing hormone and growth hormone releasing hormone)

42 The mammalian SCN SCN neurons have rhythmic firing patterns varying around a mean of 24 hr. each neuron contains a clock, but cellautonomous clocks are more variable than the behavioral clock of the whole animal. GABA and VIPmediated coupling between cells synchronizes the firing of the SCN in vivo to a precise 24 hr periodicity. periodicity of individual cells is altered by mutations that perturb the periodicity of behavior of the whole animal.

43 Synchronizing circuits within the SCN The ventral SCN receives eye input, and therefore acquires information on light. It projects densely to the dorsal SCN. These projections synchronize dorsal SCN neurons to the same periods as the ventral neurons. Neurotransmitters involved in this communication within the SCN include GABA and VIP. VIP, a peptide, may play a similar role to PDF in the fly system. The organization of the ventral SCN-- >dorsal SCN VIP circuit resembles that of the LNv -->LNd PDF circuit.

44 Peripheral cell-autonomous clocks exist in many or all peripheral tissues these peripheral clocks run 4-6 hr behind the SCN clock, probably because they are synchronized by delayed humoral outputs from the SCN. These clocks are robust, and continue to run at the same speed as cells divide, even though the concentrations of cell components are likely to change during growth and division.

45 Peripheral clocks allow circadian control of many outputs that all have different phases these outputs do not have to be separately controlled by timed release of SCN hormones. instead SCN synchronizes all body clocks by rhythmic release of a few key hormones circadian outputs from tissues are then separately regulated by clocks in their cells.

46 Resetting of the mammalian clock

47 Mechanism of resetting of the SCN clock by light The phase-shifting inputs from the retina to the SCN are mediated by light-sensitive retinal ganglion cells that express the chromophore melanopsin. Input from the retinohypothalamic tract during the night induces Per in the SCN, shifting the clock phase.

48 Clock reviews *1. Allada, R., and Chung, B.Y. (2010) Circadian organization of behavior and physiology in Drosophila. Annu. Rev. Physiol. 72: *2. Colwell, C.S. (2011) Linking neural activity and molecular oscillations in the SCN. Nature Reviews Neurosci. 12, *Required reading. For primary references for each lecture see the bibliographies of the listed reviews or ask an instructor. PDFs of reviews and papers for student-led discussions can be downloaded from the Bi156 website.

49 Papers for student-led discussions (other choices possible after consultation with profs/tas) 1. Nakamura, T. J. et al. Age-related decline in circadian output. J. Neurosci. 31, (2011). 2. Abruzzi, K.C. et al. Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression. Genes Dev. 25: (2011). 3. O Neill, J. S., Maywood, E. S., Chesham, J. E., Takahashi, J. S. & Hastings, M. H. camp-dependent signaling as a core component of the mammalian circadian pacemaker. Science 320, (2008).