SJ Wood and CR Slater. Rachel Armitage

Transcription

1 SJ Wood and CR Slater Rachel Armitage

2 Found that postsynaptic folds and the VGSCs within them doubles the safety factor at NMJs in rats in vitro

3 Background Aims Hypothesis Methods Results Conclusion Discussion Future Work Burning Question

4 At many vertebrate NMJs there are lots of post-synaptic folds Possible that high resistance pathways produced by folds focusses endplate current flow on the VGSCs in the depths of the folds This could increase release of transmitter

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6 Defined as the ratio of the normal quantal content to the number of quanta required to reach threshold The number of quanta required to reach threshold is substantially less than normally released Can be estimated by assessing both the amount of transmitter released per nerve impulse and the amount of transmitter required to initiate an action potential

7 EDL- fast twitch Soleus- slow twitch Fast twitch known to have higher safety factor: More functional Ach receptors Higher density of VGSCs in both J and XJ regions Different organisation of post synaptic folds EDL- fast twitch soleus- slow twitch

8 To investigate relationship between post-synaptic folds and transmitter release

9 Function of postsynaptic folds and VGSCs within them is to enhance efficacy of neuromuscular transmission Rat EDL (fast twitch) will have an increased enhancement of safety factor than soleus (slow twitch) due to different organisation of postsynaptic folds

10 Measure amount of transmitter released by a single nerve impulse for soleus and EDL Find the threshold for action potential generation at J and XJ for soleus and EDL Ultrastructural analysis for quantitative comparisons of soleus and EDL

11 Nerve muscle preparations were taken Action potentials blocked by μ- conotoxin μ-conotoxin is a muscle specific VGSC blocker Limitation- shown to block sodium currents in mouse nerves at high concentrations (Braga et al 1992).

12 EPP potential not entirely accurate due to non-linear relationship between amount of transmitter released and resulting depolarisation Each rise in conductance represents a smaller and smaller fraction of the total conductance as the number of quanta increases

13 To avoid underestimation of quanta, Martin s non-linear summation can be used from EPP and MEPPs Limitation- capacitative properties of muscle fibre also influences EPP. Efforts have been made to account for this but still not entirely accurate

14 Provides direct estimate of transmitter release Recorded currents are linearly related to the number of ion channels opened by released Ach EPC and MEPC best method available Limitation- effectiveness of voltage regulation decays rapidly with distance from the electrodes

15 Action of Ach released from the nerve used to determine threshold Δ-tubocurarine partially blocks response of the muscle to Ach so that occassional NMJs can be found where the amplitude of the EPP is close to threshold Complications- current and voltage electrodes must be placed close to the NMJ to minimise effects of spatial decay of applied current and voltage

16 Use 2 intracellular electrodes Pass rectangular pulses of current through one while recording resulting changes with the other Measures smallest abrupt depolarisation Performed on J and XJ region Not very representative of vivo

17 Calculated in terms of charge (more accurate) Calculated in terms of voltage to allow comparisons to other studies

18 Light microscopy Electron microscopy

19 Quantal content in EDL (fast twitch) 30-40% higher than in soleus P<0.001 with t tests

20 Method 1 both soleus and EDL muscles require about 13 quanta to be released in order to reach threshold Method 2- Threshold times higher than nerve evoked Amount of charge required to reach threshold 20% less at J than XJ in soleus and EDL EDL (fast twitch) required approx 15% more charge to reach threshold than those in soleus at both J and XJ regions Depolarization required to reach threshold was significantly lower at J than XJ in soleus but not in EDL

21 Safety Factor in terms of charge by nerve evoked activation was 3.5 in soleus and 5.0 in EDL which is double safety factor of XJ area in both cases Safety factor in terms of voltage by nerve evoked activation was 3.1 in soleus and 3.4 in EDL which is again approx double safety factor of XJ area

22 Soleus muscle fibre diameter and area of NMJs 50% bigger therefore ratio is the same Extent of postynaptic folding- fold index represents 5 fold increase in amount of membrane at NMJ in both soleus and EDL EDL releases x2 transmitter per unit area of synaptic contact than soleus. Suggests increases transmitter release accounts for greater safety factor in EDL

23 Post-synaptic folds cause a 2 fold increase in safety factor in rat EDL and soleus Safety factor of EDL is greater than that of soleus. Amount of membrane per fold is the same in both muscles EDL releases x2 transmitter per unit area of synaptic contact than soleus

24 -Differences from in vivo: -temperature (transmitter release increases as temperature increases)- might suggest safety factor would be higher at 37 C than temp used here (21 C) -Pattern of impulse activity in motor nerves: -Soleus- long 20Hz bursts whereas EDL short Hz bursts -Quantal content at NMJ is reduced as frequency and duration of stimulus is increased. This suggests safety factor might be reduced in vivo, particularly for soleus.

25 Previous studies found more extensive folding in fast twitch- not quantified. Could be due to different muscles being used. (respiratory vs leg muscles)

26 Compare NMJs with different amounts of folding Test hypothesis in humans using different AP blocker

27 A number of forms of inherited myasthenic syndrome have been identified where a common feature is reduction in extent of postsynaptic folding Possible congenital paucity of secondary synaptic folds syndrome

28 Presynaptic nerve terminal size- (increase) pattern of activity- pattern activation of motor units in vivo is very different from that used in most estimates of safety factor in vitro. See above about long low frequency and short high frequency. short term modulation of release- quantal release from motor nerve terminal can be modulated by activation of receptors located on presynaptic terminal or schwann cells. cholinergic modulation- purinergic modulation adrenergic modulation Postsynaptic features: AChE- decreases density and distribution of AChRs- known to occur in a number of pathological situations VGSCs/ folds- lowers threshold muscle fibre diameter and Rin- smaller muscle fibres require fewer quanta to reach threshold than larger ones- not particularly important for safety factor- adaption by nerve terminal?

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36 Active Zones and the Readily Releasable Pool of Synaptic Vesicles at the Neuromuscular Junction of the Mouse Ruiz, R. Cano, R. Casanas, J.J. Gaffield, M.A. Betz, W.J. and Tabares, L. (2011) The Journal of Neuroscience. Vol. 31, Issue 6, pp Artemis Zormpa

37 Background Posi&onal Priming : a0er a vesicle from the RRP undergoes exocytosis it is replaced by another nearby vesicle

38 Active Zones 2 primary docked vesicles 2 3 secondary docked vesicles Tethered vesicles High density of calcium channels Molecules media&ng docking, fusion and clearance of synap&c vesicles

39 Aim To determine the size and properties of the readily releasable pool of synaptic vesicles using both electrophysiological recordings and morphological measurements.

40 Methods Levator auris longus muscle from mice of both sexes Immunostaining (Bassoon and Piccolo label scaffolding proteins of the AZ) Confocal microscope imaging Electrophysiology

41 Morphological measurements (1) No correla&on between diameter of AZs and amount of fluorescence No specific loci in terminal for max fluorescence Average size of AZs was ~ 300nm No of AZ propor&onal to terminal size ~ 850 AZs per terminal (x2 primary vesicles) = ~ 1700 vesicles (+2ry vesicles)

42 Morphological measurements (2) AZs had a fixed spa&al pawern Nearest neighbor was found at ~ 530nm distance Every AZ was found on top of a postsynap&c fold Spa&al pawern disrupted only by proteoly&c enzymes and NOT through massive exocytosis

43 Electrophysiological recordings (1) EPPs decrease gradually un&l the reach a plateau So does the quanta contents (m) The greater the interval between s&muli the greater the RRP recovery

44 Electrophysiological recordings (2) At higher s&mula&on frequencies: Quantum content decreases faster Plateau amplitude decreases Rate of deple&on increases Rate of refilling increases RRP size increases Some AZs are not ac&ve at low frequencies

45 Electrophysiological recordings (3) T PE EGTA Initial amplitude Plateau amplitude Rate of depletion Rate of refilling no yes yes yes yes yes no no yes yes yes yes RRP size yes no yes Quantum content yes yes yes

46 Electrophysiological recordings (4) Dead &me (=#me required a-er exocytosis for a vacant vesicle site to be refilled) decreases exponen&ally with increasing s&mulus frequency Number of release sites increase with increasing s&mulus frequency

47 Summary (1) AZs are found over postsynaptic folds RRP released first and newly recruited vesicles reach a plateau with endocytosis Rate of recruitment is almost inversely proportional to rate of refilling Plateau indicated no change in recruitment pattern (ie. Reserve pool or endocytosis?)

48 Summary (2) RRP size changes at different stimulation frequencies (sleepy AZs calcium dependent) Refilling is faster during electrical stimulation than at rest (due to calcium concentration) Temperature and PE increase rate of recruitment EGTA decreases rate of recruitment

49 Evaluation No sample size randomization age Many assumptions (eg. Constant dead time, dead time duration, etc.) No suggestions for further research and implications of findings Not clear whether plateau was reached due to endocytosis or use of the reserve pool Proved the existence of sleepy AZs Gave insight into mechanisms of exocytosis Mechanism of how higher stimuli produce a greater response

50 Further Research More muscle types (possible differences in fast- and slowconducting nerves?) Visual characterization of active zones involved in exocytosis (maybe a pattern? synaptophluorin/time-lapse) Quantifying endocytosis to verify some of the assumptions (FM dyes) Quantifying reserve pool before and after stimulation (using synaptophluorin/time lapse) Generally more morphological measurements (at all stages) Quantification in motor neuron disease, myasthenias, channelopathies, etc.

51 Big Burning Question What insights does this paper give into the relationships active zones, quantal content, exocytosis and vesicle replenishment? E.g Is there a hierarchy of active zone recruitment?

52 Preferred Sites of Exocytosis and Endocytosis Colocalize during High- But Not Lower-Frequency Stimulation in Mouse Motor Nerve Terminals Michael A. Gaffielf, Lucia Tabares and William J. Betz Gemma Stevenson

53 Exocytosis Gundelfinger et al, Access science

54 Endocytosis A Rodal et al, 2008 K. Powell, 2003

55 Hypothesis Hot spots of endocytosis, possibly in the form of bulk uptake, occur at or very near highly active exocytic sites during high-frequency stimulation Aim The authors sought to address the degree of colocaliza&on of exocy&c and endocy&c sites in real &me in living motor nerve terminals during different s&mula&on intensity levels

56 Previous studies Spatial locations of endocytic sites have been proposed to emerge in close proximity to exocytic sites at the NMJ (Roos and Kelly, 1999; Teng and Wilkinson, 2000)

57 Experiment design Real-time experiment to determine spatial properties using synaptophluorin (sph) and FM4-64 sph hot spots provide exocytic sites FM dyes also can show exocytic sites, but due to their unquenchable properties determine endocytic sites. were able to report FM4-64 spots arise close to sph spots during intense but not moderate stimulation

58 Methods Muscle preparation sph and FM4-64 dye loading Image analysis, stimulation and modeling Statistical tests

59 FM4 64 during 100Hz s&mula&on reveals spots (figure 1)

60 FM4 64 and sph spots have similar characteris&cs (figure 2)

61 FM4 64 spots disperse slowly a0er s&mula&on (figure 3)

62 Colocaliza&on (figure 4)

63 Quan&ta&ve results, 100Hz (Figure 5)

64 Spa&al Proper&es at moderate s&mula&on (figure 6)

65 Quan&ta&ve results for high and moderate s&mula&on (comparing figure 7 and figure 5)

66 Conclusion FM4-64 spots arose close to sph spots during intense but not moderate stimulation From the data, individual hot spots were found to be randomly scattered throughout the terminal but in close proximity to each other

67 Limitations Number of spots tested, trials and repeat experiments were sometimes limited. In the nearest-neighbor experiments, some data was not shown why? In the comparison of figure 7 and figure 5, due to the absent of some data it was not able to compare the lower frequency to higher frequency data of individual spots

68 Further Work Rather than focusing on bulk uptake endocytosis, could specifically look at colocalization between clathrin-dependent endocytosis and exocytosis sites. Used non-physiological stimulation durations, would be interesting to see if any colocalization at physiological duration Repeat the experiments at varying temperatures Spatial analysis of other endocytosis properties. The exceptions of colocalization could also be investigated further.

69 Big Burning Question Why does the relationship between exocytosis and endocytosis sites change at different frequencies?

70 References A. A. Rodal, J. T. Littleton. Synaptic Endocytosis: Illuminating the Role of Clathrin Assembly. (2008) Current Biology., 18: D. Gundelfinger, M. Kessels & B. Qualmann. Temporal and spatial coordination of exocytosis and endocytosis. (2003) Nat. Rev. Mol. Cell. Biology 4, K. Powell. Viruses catch and endocytic ride into the cell. (2005) J. Cell Biol., 88: Synaptic transmission. Access Science McGraw Hill D= (accessed on 18 October 2012)

71 Pre and post synaptic abnormalities associated with impaired neuromuscular transmission in a group of patients with limb-girdle myasthenia Slater CR, Fawcett PR, Walls TJ, Lyons PR, Bailey SJ, Beeson D, Young C, Gardner- Medwin D. Jessica Brohier

72 Contents Background Methods Results Discussion Big Burning Question Critical Appraisal

73 Background Myasthenic Syndromes Conditions causing abnormal weakness of certain muscles. Myasthenia Gravis Lambert Eaton Syndrome Congenital Myasthenic Syndromes

74 Limb-girdle Myasthenia (LGM) Rare Disorder of impaired neuromuscular transmission Weakness of proximal limb muscles Little or no involvement of ocular or facial muscles Characteristic waddling gait

75 Aims Identify any structural and functional abnormalities of the NMJ in patients with LGM. Determine if the defects are pre or post synaptic

76 Methods 8 LGM patients selected based on clinical examination 8 control patients selected with idiopathic muscle pain or adult onset muscular dystrophy

77 Intracellular recording Motor point biopsy samples obtained from Vastus Lateralis (VL) Intracellular recordings performed using microelectrodes to record membrane potential. Two-electrode voltage clamp configuration was used to record end plate currents (EPC) Subsequent computer analysis measured EPCs and calculated quantal content (QC).

78 Imaging techniques Acetylcholinesterase (AChE) activity demonstraited histochemically AChRs fluorescently and radio labelled with α- bungarotoxin (α-bgtx) conjugates. Light microscopy Electron microscopy Immunocytochemistry- Antibody labelling to detect abundance and distribution of postsynaptic proteins

79 DNA Analysis Genomic DNA from LGM and control samples were analysed for mutations in genes commonly associated with congenital myasthenia: AChR subunit genes (CHRNA, CHRNB, CHRND, CHRNE), RAPSN, COLQ, CHAT and MUSK.

80 Results

81 Electrophysiolog y Intracellular recordings show significantly impaired neuromuscular transmission Mean EPP amplitude of LGM pa&ents only 46% that of controls

82 Quantal content amount of neurotransmiwer released Computer analysis revealed reduced quantal content in LGM samples compared to controls. Mean quantal content was 59% of the control value

83 Light Microscopy LGM pa&ents NMJs appear smaller and less compact (A,B) No significant difference in abundance and distribu&on of AChR between control and LGM (C,D) Synap&c area, as determined by area of ChE ac&vity, in LGM only 50% that of controls (E,F)

84 Electron microscopy Reduced postsynap&c folding in LGM pa&ents (C,D) compared to controls (A,B) Varia&on in extent of folding present in the same biopsy sample (F,G)

85 Synap&c area (determined by ChE ac&vity) was shown to correlate with quantal content Average ra&os of quantal content/synap&c area showed the same release of quantal content per area of synap&c contact as control samples

86 Fold index is a measure of the increase in postsynap&c length caused by folding Fold index= Length of folded membrane/ Surface length This was found to be 50 60% of the control values in LGM samples.

87 Immunocytochemic al Labelling An&body labelling showed the presence of all 13 proteins at the NMJs of LGM and control pa&ents. 14 proteins were analysed in total (Neuregulin 1 not pictures) and all showed normal distribu&on and abundance.

88 DNA Analysis No mutations were found in any of the genes commonly associated with congenital myasthenic syndromes: AChR α, β,δ and ε subunits, rapsyn, ColQ, cholinacetyltransferase and MuSK.

89 Discussion The main finding of this study indicate that the abnormality lies in reduced synap&c contact at the NMJ and not simply reduced quantal content. This reduced synap&c contact is though to be due to abnormali&es a that are both pre and post synap&c Light microscopy showed that the overall size of the NMJ is reduced in limb girdle myathenia pa&ents. Electron microscopy showed that the post synap&c membrane exhibits reduced folding resul&ng in increased threshold for muscle fibre ac&on poten&al genera&on

90 Immunocytochemical Labelling and DNA analysis ruled out a number of protein mutations as a cause for the reduced synaptic transmission observed in LGM The observed abnormalities in NMJ structure have highlighted an important area of research in the underlying mechanism of this disorder

91 Big Burning Question What is the relationship between the cellular phenotype and the clinical presentation in CMS/LGD? Why are there reduced folds?

92 What is the relationship between the cellular phenotype and the clinical presentation in CMS/LGD? Reduced folding of the post-synaptic membrane of the NMJ results in impaired neuromuscular transmission.

93 Why are there reduced folds? A homozygous mutation of the gene coding Dok-7 results in reduced synaptic folding in Limb-girdle myasthenia patients (Palace & Beeson 2008). Dok-7 is an adaptor protein essential for neuromuscular synapse formation.

94 Dok7 and MuSK interac&on Dok7 PTB domain which interacts with MuSK is mutated in some congenital myasthenias Interac&on between Dok7 and MuSK is essen&al for NMJ forma&on

95 Critical Appraisal Study performed on human pa&ents which has advantages and limita&ons...

96 Strengths Study performed on human therefore directly relates to the human condition and treatment Very extensive study, many aspects of structure and function explored Imaging techniques found clear abnormal cellular phenotype

97 Limitations Experiments performed on muscles from different locations Limited muscle samples available Large variation in the extent of folding present within the same biopsy sample results in large standard deviation making statistical analysis difficult and unreliable Small sample size with only 8 patients and 8 control patients

98 Future Work Screen for mutations of other genes involved in the formation of the NMJ Use of Dok-7 mutant mice as model for Limb-girdle myasthenia