R-15. How does it work? What does it do? Does anybody still care? Even if not, was it all worth it?

Transcription

1 R-15 How does it work? What does it do? Does anybody still care? Even if not, was it all worth it?

2 Aplysia Genus Aplysia that which does not wash Gmelin, 1789 studied by naturalists in 1800s molluscan biologists in early 1900s (Eales) ideal simple organisms to study the cellular basis of behavior (Kandel) (photo: wikipedia commons)

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5 R15 Early days of intracellular sharp microelectrode recording Arvanitaki and Chalazonitis (1955). Les potentials bioélectriques endocytaires du neurone géant d Aplysia en activité autorhytmique. Tauc (1954) Réponse de la cellule nerveuse du ganglion abdominal d Aplysia depilans á la stimulation directe intracellulaire.

6 These two turned Eric Kandel onto Aplysia... In Search of Memory: The Emergence of a New Science of Mind, E. Kandel

7 In Search of Memory: The Emergence of a New Science of Mind, E. Kandel

8 The first bursting neuron studied? Strumwasser (1968) - parabolic bursting

9 An endogenous burster Early studies could find no evidence of rhythmic synaptic input or synchronous activity in other neurons Bursting could be phase-shifted by welltimed inputs (Pinsker, 1977) Isolated somata can burst (Alving, 1968, via ligature; Chen et al., 1971, via dissociation)

10 How does it work? i.e. how does it burst? The generation and modulation of endogenous rhythmicity in the Aplysia bursting pacemaker neurone R15. WB Adams and JA Benson Prog. Biophys. Mol. Biol. (1985) A comprehensive review of ionic mechanisms, no known papers published since then on ionic mechanisms underlying bursting!

11 Heavily studied in 1970s and early 80s Early recognition of the importance of a negative slope region in the I-V curve correlating with the ability to burst Recognized that slow conductance changes in inward and outward currents occurred during the burst Which ions, and what is slowly changing?

12 Junge and Stephens, 1972

13 Junge and Stephens, 1972

14 chaos of conflicting observations and interpretations (Adams and Benson, 1985) Some could obtain a subthreshold oscillation with TTX, others could not Other criticized much of this work, citing wholesale removal of Na or Ca as resulting in highly abnormal changes in membrane resistance Fresh TTX blocks I NSR, but not 1 day after opening vial (Futamachi and Smith, 1982) To this day, not nailed down. Ca or Na or both.., but all recognize NSR is responsible for triggering burst

15 What terminates the burst? 1970s - slow activation and inactivation of a K+ current (Dan Johnston) 1980s - V-act and slow Ca-inact of the NSR current (Adams and Benson and Gage, Kramer and Zucker) Adams (1985) - IH summates during the burst and helps to terminate bursting, but it is active throughout the burst cycle due to a long time constant...

16 Unsolved We now know, due to the complexity of other well studied systems (Ex. leech) that the basis of bursting may be a complex combination of factors. Most models since 1980s rely on a voltageactivated ca-inactivated inward current to start and stop the burst... (plus more) Hard to measure these currents - many subthreshold currents are simultaneously active

17 Why is this unsolved? Other systems got more interesting 1980s - patch clamp Rise of mammalian physiology There seems to not even be a good physiological reason to study bursting in R15!

18 How to publish your PhD Chronic recording of R15 activity in vivo indicates that it does not burst spontaneously in the intact animal.

19 1-4 days chronic recording 1-2 days after surgery

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21 NIH CRISP search last funded EXPERIMENTAL studies by NIH on R15 Aplysia and bursting : John Koester (ended 1995) - funded the work just mentioned... Aplysia and R15 : same, Jack Byrne (but was a modeling study linking genes to membrane dynamics)

22 We could figure it out... We don t know how it burst We COULD - modern genetics and molecular biology, Aplysia genome project, knowledge of ion channel families Adams/Bensons descriptions of ion channels were more functional than specific Would NIH fund it today? The heavily studied with lots of data argument isn t competitive anymore...

23 Major IMPACT The discoverers of R15 trained Kandel, who trained many others... Computational models of bursting Methods for studying the dynamics of bursting systems (fast/slow analysis) motivated by R15 and pancreatic beta cells Circadian rhythms - Strumwasser (1965) noted long term changes in bursting during prolonged intracellular recordings Light and temperature sensitivity Phase-Resetting Curve methods (Pinsker, earliest known PRCs measured from bursting neurons) Neuromodulation

24 Neuromodulation Some of the earliest and most complete work showing: Neuromodulators can act on multiple ion channels with separate dose-responses These actions can occur directly or indirectly Indirect effects mediated by second messengers

25 This paper pulls together about 10 years of study of how 5-HT mediates R15 activity

26 control 10 um 5-HT washout 50 um 5-HT

27 control low doses of 5-HT hyperpolarize 10 um 5-HT the interburst (and sometimes silence the cell) washout high doses depolarize and enhance the burst itself, and often lead to a transition to 50 um spiking 5-HT

28 Dose effects on I-V curve

29 5-HT turns on non-brief responses to brief inputs control various 5-HT concentrations

30 A model

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35 Even this is incomplete... (ignores Ca/Calmodulin interactions with camp pathway)

36 This model made a lot of assumptions, but suggested Yu, Byrne, and Baxter, 2004 possibilities...

37 Undone Yu s model had a lot of assumptions on rate constants (easier to measure eq. constants) +/- interactions b.w. camp and Ca suggest that metabolic oscillations are possible, with or without the membrane dynamics A candidate model cell for studying regulation of ion channels by modulators, long term effects, endogenous activitydependent gene expression?

38 Models are not as versatile as data Features of burst vary greatly No model ever exhibits the well-sculpted mode of bursting to the extent of the real neuron No model exhibits oscillations in repetive spiking

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