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Outline

Outline. Special synapses and preps Methods for looking at release Molecules of vesicle fusion. Calyx of Held. Auditory brainstem structure involved in localization of sound HUGE presynaptic terminal makes it possible to patch Calyx-like synapses usually seen when failure isn’t an option.

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Outline

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  1. Outline Special synapses and preps Methods for looking at release Molecules of vesicle fusion

  2. Calyx of Held • Auditory brainstem structure involved in localization of sound • HUGE presynaptic terminal makes it possible to patch • Calyx-like synapses usually seen when failure isn’t an option

  3. Microcultures Allows analysis of neurons that have been modified to affect presynaptic processes

  4. Response = NPrQ N = number of vesicles or synapses Pr = probability of release can depend on [Ca2+] influx, sensitivity or vesicle priming Q = quantal size amount of NT in a vesicle, or postsynaptic response to a vesicle

  5. Ways to change NPrQ • N = number • synaptogenesis; imbalance endo-exocytosis • Pr = probability of release • change [Ca2+]; influx; exocytotic molecules • Q = quantal size • transporter expression; receptor expression

  6. Methods for measuring presynaptic activity • Electrophysiological recordings • Miniature and evoked PSCs • Capacitance recordings • Amperometry • Imaging • Calcium sensors • FM styryl dyes

  7. Thompson et al., 1993 Miniature EPSCs sEPSCs- “spontaneous EPSCs” are a mixture of AP evoked and miniature EPSCs recorded in absence of stimulation due to random firing of neurons mEPSCs- “miniature EPSCs” are events in the presence of TTX (not due to APs) due to the calcium independent fusion of single vesicles Changes in mini frequency are due to changes in N or Pr Changes in amplitude are due to changes in Q

  8. Dodge and Rahamimoff, 1967 Evoked EPSCs Evoked by stimulating a bundle of fibers, a presynaptic cell, or a presynaptic terminal

  9. Cooperativity Usually indicates more than one binding site Binding sites are NOT independent

  10. Mean-Variance Analysis Murphy et al., 2004

  11. Paired Pulse Ratio (PPR) PPR = Pulse2/Pulse1 Result of the balance between facilitation and depression Facilitation- a calcium dependent process where more probability of release is increased on a second pulse Depression- a decrease in probability of release on a second pulse due to vesicle depletion (indicative of high Pr)

  12. Capacitance Recordings IR = V/R IC = C(dV/dt)

  13. Amperometry

  14. Calcium Imaging Two classes of indicators: synthetic dyes and engineered proteins Dyes that shift emission wavelengths allow ratiometric analysis to compute [Ca2+] Synthetic dyes can be loaded into a single cell via patch pipet or applied to entire prep DNA encoding calcium sensing proteins can be incorporated into the genome

  15. Calcium Imaging Issues Compromise between speed and buffering Bleaching Localization

  16. Calcium Uncaging Activation by UV light can change they shape of a molecule by breaking bonds This decreases Calcium affinity Many compounds can be caged with similar techniques

  17. FM-Styryl Dyes Klingauf et al., 1998

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