Dimorphism and Homeostasis of Neuromuscular Synaptic Function

Transcription

1 Dimorphism and Homeostasis of Neuromuscular Synaptic Function - Epp m ε Release + + φ - q Sensitivity The nature of the problem Size, Strength, Resistance.. The Power of Drosophila The nature of the problem Size, Strength, Resistance.. The Power of Drosophila 1

2 Homeostasis (Walter B Cannon, 1932) The maintenance of physiological variables within closely confined limits. Classic examples: core temperature; blood glucose* *Normal range (Set Point): mm Hyperglycaemia (>10mM) --- > Insulin --> Glucose uptake Hyoglycaemia (<4 mm) ---> Glucagon (+others) --> Glucose release Typically, different mechanisms compensate for positive and negative deviations from the set-point Shiver 37 0 C Sweat Glucagon 5mM Insulin Core Temp [Glucose] - + O E Uptake ε Insulin + - Release Glucagon V Voltage clamp ~ ±I Desaki & Uehara,

3 AC mv Transferred Traces abf V I Transferred Traces abf V Φ V =V E /V t V E V t Wood & Slater (1997) Desaki & Uehara, 1981 NMJ operate with a high safety factor 100mN Ribchester (1993) J Physiol Ruiz et al (2007) J Neurosci 3

4 Short-term regulation of quantal content Ruiz et al (2011) J Neurosci Safety factor is maintained at high frequency 100Hz Φ q Wood & Slater (1997) Firing threshold is lowest at the motor endplate Wood & Slater,

5 Non-linear summation weakly buffers safety factor Φ V =V E /V t V E V t Wood & Slater (1997) Φ q =m/m t m=v / q V =V/(1-fV/E 0 ) E 0 =(E m -E r ) Φ V =V/(E m -E t ) f=0.8 q=1mv E m =-75 mv E r =-5 mv E 0 =70mV E t =-65mV Working Hypothesis - Epp m ε Release + + φ - q Sensitivity The nature of the problem Size, Strength, Resistance.. The Power of Drosophila 5

6 NMJ size and muscle fibre diameter co-vary Kuno et al., 1971 NMJ size and muscle fibre diameter co-vary, even in diseased muscle 20 µm R6/2 Wild type Fibre Diameter (µm) Ribchester et al. (2004) Eur J Neurosci The size of NMJ and the extent of junctional folding vary between species Frog Frog Rat Rat Man Man 6

7 %Occupancy Evoked release and NMJ area are correlated 200 Frog Synaptic area (µm 2 ) Rat Human EPP amplitude is proportional to fractional occupancy of endplates RH414/FM µm 10 ms Costanzo et al.(1999) J Physiol 521: Specific quantal content (m/µm 2 ) is roughly constant A B ) mµ m/a (µm -2 ) 0.1 m/a ( sub super sub-sub sub-super super-super Costanzo et al.(1999) J Physiol 521:

8 Vm 2.5 mv Ch.2 1 mv ms Why is a relationship between endplate size and muscle fibre size important? Determinants of EPP amplitude EPP amplitude = I s * R in I s (TR, #Receptors) R in (Rm, Size -1 ) Slide courtesy of Greg Lnenicka, SUNY Albany R m =!dr m " R i =! d % # $ 2 & ' 2 r i R in = 1! R m.r i d 3 r i r m R in! 1 d 3 d R in =!V!I 8

9 Vm 2.5 mv Vm Ch mv 1 mv Ch.2 31 Keyboard AC mv mv Ch.2 10 mv ms ms ms s Vm 2.5 mv Ch.2 1 mv ms AC mv Ch.2 10 mv 5.00 ms s 1 2 R in (MΩ) Harris & Ribchester (1979) J Physiol Synaptic size-strength regulation compensates for diameter/input resistance nt mf R in = 1 R m R i A! d 3 t! d m V 10 mv 2 na I = R in 20 ms q! R in MEPPs m! A t EPPs Ribchester et al., 2004 Eur J Neurosci Determinants of safety factor (Φ V ) Quantal content (m; presynaptic) - Probability of transmitter release (p; Ca 2+ ) - Active zone number and docked vesicles (n) -Transmitter store size and mobilisation - Nerve terminal size Φ V = (mq) /(E t -E m ) Quantal size (q; postsynaptic) - Vesicle filling - Cholinesterase activity - ACh receptor density; Reversal potential (E r ) - Muscle fibre diameter and input resistance - Junctional fold density (Na channel density-->e t ) 9

10 Working Hypothesis - Epp m ε Release + + φ - q Sensitivity Quantal size can be varied by: -vesicle content of neurotransmitter -Rate of loss of neurotransmitter (AChE, diffusion, uptake) -Density of postsynaptic receptors -Input resistance of muscle fibre -Density of postjunctional folds Quantal content can be varied by: n -Number of active zones (density; terminal area) -Number of docked vesicles -Rate of mobilization of undocked vesicles -Efficiency of vesicle recycling p -Ca influx -Ca buffering/removal -Sensitivity of Ca sensors -Efficiency of exocytosis 10

11 Myasthenia gravis and LEMS are autoimmune diseases LEMS: Ca channel antibodies X X X X MG: AChR antibodies Assembly of the motor endplate depends on DOK-7 11

12 Area-diameter relationships break down in DOK-7 mutations that produce CMS Slater et al. (2006) Brain Consequently,synaptic strength, but not quantal size, is reduced in CMS How might Safety Factor be homeostatically regulated? Epp = f(m,q,e m -E r ) m = np n = f(a z,v d, V m, V r ) p = f([ca 2+ ]) q = f(v f,ache,achr,r in ) E t = f(j f,nach) φ = f(epp,e t ) 12

13 Proteins of the Active Zone Neuromuscular Junction: postsynaptic The nature of the problem Size, Strength, Resistance.. The Power of Drosophila 13

14 The Fly Little Fly, Thy summer's play My thoughtless hand Has brushed away. The Power of Drosophila Vitruvian Man - Leonardo da Vinci Am not I A fly like thee? Or art not thou A man like me? For I dance And drink, and sing, Till some blind hand Shall brush my wing. If thought is life And strength and breath And the want Of thought is death; Then am I A happy fly, If I live, Or if I die. Images and design courtesy of Giusy Pennetta William Blake ( ) Human chromosomes 3.1 billion base pairs: ca. 20,050 genes (proteins) million base pairs : ca. 15,016 genes 14

15 Metaphase chromosomes in Drosophila About 50% of fly protein sequences have mammalian homologs and about 75% of known human disease genes have a recognizable match in the genome of fruit flies. 15

16 Synaptic degeneration in the glued (dynactin) mutant Eaton et al (2002) Dynactin is necessary for synapse stabilization. Neuron 34, Homologous mutations in glued (dynactin) are a cause/risk factor for ALS 16

17 Song et al., 2007, PNAS Drosophila 3rd Instar fillet 17

18 Each half segment contains 30 muscle fibres supplied by 30 motor neurones Hoang & Chiba (2001) Most muscle fibres are polyinnervated 18

19 Drosophila NMJs Muscle fibre Axon Type II boutons Type I boutons Nerve terminals bind peroxidase antibodies There are three main types of boutons or varicosities: Type Ib (b= b= big ), Type Is (s= s= small ) and Type II. Glutamate is the excitatory neurotransmitter mainly released by the type I boutons. Peptides such as octopamine are released by the type II boutons and they may have a modulatory role. Drosophila NMJ - Scanning EM: Nerve terminal is embedded in the muscle surface Yoshihara et al (1997) J Neurosci 17, Pielage et al. (2011)PMID m = n.p n ---> active zones p---> [Ca] i Jiao et al. J Struct Biol PMID

20 The larval NMJ is accessible to electrophysiological analysis J Neurobiol May;43(2):

21 Drosophila Fibre 5, segment A4 10 µm Sara Al-Najar/RRR 21

22 Drosophila Fibre 5, segment A4 Sara Al-Najar/RRR Drosophila Fibre 5, segment A4 NC82+ puncta associate with membranes Sara Al-Najar/RRR Female : Third instar Drosophila Larva Fibre 5: 10 Hz Stimulation Pseudocoloured Surface Plot Greg Lnenicka 22

23 CSP In CSP Southall et al., 2008 Drosophila 3rd Instar Larva : Motor Unit VNC Soma diameter ~ 10 µm : Volume ~ 500 µm 3 Axon length ~0.3-3 mm : Volume ~ µm 3 NMJ length ~ 100 µm x 3 = 100 boutons: Volume ~ 500µm 3 OK6-Gal4:UAS-myr-RFP 20 µm ~1 mm VNC T Mosca, T Schwartz & RR Ribchester (on his sabbatical ) Segment A3: fibre 7/6/ 13/ 12 NMJs Segment A5: fibre 6/7 NMJ Davis & Goodman, 1998 Nature 392,

24 Frank et al.(2006) Neuron 52, Increased sensitivity retargets set point muscle excitation Set point muscle excitation Impair neurotransmitter release Davis, Ann Rev Neuro :

25 Chronic Mf Size TR #Receptors DeRosa and Govind (1978); Lnenicka and Mellon (1983) Nt Size TR #Receptors Davis and Goodman (1998) R m TR Paradis S, Sweeney ST, Davis GW (2001) #Receptors TR Petersen SA, Fetter RD, Noordermeer JN, Goodman CS, DiAntonio A (1997); Davis GW, DiAntonio A, Petersen SA, Goodman CS (1998); Acute DiAntonio A, Petersen SA, Heckmann M, Goodman CS (1999) #Receptors TR Frank CA, Kennedy MJ, Goold CP, Marek KW, Davis GW (2006) J Neurobiol Apr;66(5): Three key sex-determining genes: sex-lethal; transformer; double-sex Gilbert SF. (2000) Developmental Biology. 6th edition.sunderland (MA): Sinauer Associates;

26 Working Hypothesis - Epp m ε Release + + φ - q Sensitivity How might EPSP amplitude be homeostatically regulated? Epp = f(m,q,e m -E r ) m = np n = f(a z,v d, V m, V r ) p = f([ca 2+ ]) q = f(v f,ache,achr,r in ) E t = f(j f,nach) φ = f(epp,e t ) Time flies like an arrow; Fruit flies like a banana - Groucho Marx 26

27 SUMMARY The safety margin for neuromuscular transmission (excess of postsynaptic depolarisation over requirement) appears normally to be regulated within closely confined limits. Neuromuscular diseases such MG, LEMS and CMS may be considered failures of synaptic homeostasis. Safety factor is determined by several pre-and postsynaptic properties, affecting quantal content and quantal size The molecular mechanisms of synaptic homeostasis may be accessible in Drosophila, thanks to its rapid and prolific breeding; the innervation of specific identified muscle fibres by specific identifiable motor neurones; and the power of molecular genetics in this species. 27