PHYSIOLOGY AND PATHOPHYSIOLOGY OF NEUROMUSCULAR TRANSMISSION

Transcription

1 PHYSIOLOGY AND PATHOPHYSIOLOGY OF NEUROMUSCULAR TRANSMISSION Active zones and the readily releasable pool of synaptic vesicles at the neuromuscular junction of the mouse Aim: To gain more information on factors affecting the exocytosis of vesicles zones and the refilling by recruitment of the RRP at AZs. Methods: Immunohistochemical staining to identify AZs and develop a morphological estimate for RRP. Electrophysiological techniques to obtain measurements for m (quantal content). The development and use of a kinetic model of vesicle release and replenishment to determine various characteristics of neuromuscular transmission. Results and Conclusions: Staining results showed that AZs located were directly opposite postsynaptic folds. Morphological and electrophysiological estimates (using the kinetic model they developed) for the size of the RRP were similar. The model is divided into two components the release of quanta from the initial RRP (those docked before the stimulus train was initiated) and the release of quanta that had been recruited to AZs after the initial stimulus train. Higher frequencies of stimulation increased the rate of RRP depletion. The recovery of the RRP was found to have an exponential time constant of 823ms to recover to 83% of the control level. The dead time was estimated (the time between the release of one vesicle from an AZ and the docking of a new recruited vesicle), and the implications for p (probability of vesicle release) are discussed. The contribution of postsynaptic folds to the safety factor for neuromuscular transmission in rat fast- and slow-twitch muscles Aim: To calculate the safety factor of two different muscles (EDL and soleus) and consider the different components of NMJ transmission that contribute to the safety factor Methods: Using two methods to calculate EPPs (nerve stimulation and postsynaptic current injection), the normal quantal content and the threshold quantal content (the minimal quantal content to produce an EPP) for each muscle was calculated. The difference in charge required to generate an EPP at the NMJ (J) and outside the NMJ fold specialisations (XJ) was calculated. The safety factor was then calculated as the normal quantal content divided by the threshold quantal content. The structure of the NMJs was investigated using light and electron microscopy.

2 Results and Conclusions: The quantal content in the EDL (fast-twitch) was 40% higher than that in the soleus (slow-twitch). The safety factor in the EDL was slightly higher than in the soleus, but the difference in safety factor between J and XJ regions on the muscle fibre were the same for both muscles the safety factor was 2 times higher in the region containing postsynaptic junctional folds than at other regions on the muscle fibre. It therefore seems that the effect of the postsynaptic folds is to double the safety factor. This is due to the increased resistance within the thin strips of cytoplasm in the fold, which enhances the depolarization lower in the folds, where there is an abundance of voltage-gated sodium channels. This high safety factor is especially important in extended periods of stimulation, and maintains the reliability of neuromuscular transmission. Severely impaired neuromuscular synaptic transmission causes muscle weakness in the Cacna1a-mutant mouse rolling Nagoya Aim: To investigate the effects of the missense mutation in the Cacna1a gene encoding neuronal Ca v 2.1 on ACh release at the NMJ. Methods: Physical assessments of muscle function in RN mice (grip test and inverted grid hanging test), electrophysiological studies of the NMJ using different drugs to manipulate MEPPs and EPPs, morphological studies of NMJ and muscle fibre size from alpha-bungarotoxin binding. Results and Conclusions: RN mice showed weaker grip strength and shorter grid hanging times. Electrophysiological studies showed that they had a reduction in evoked ACh release which was due to the effect on the Ca v 2.1 channel. RN mice also had increased spontaneous ACh release, suggesting perhaps a difference between low-voltage and high-voltage activated channels. The mice seemed to have a number of shared features with the human pathology LEMS, in which circulating antibodies block presynaptic Ca v 2.1, including increased synaptic facilitation and decreased rundown. A genedosage dependency effect was found in a number of different variables by investigating heterozygous and homozygous RN mice.

3 Pre- and post-synaptic abnormalities associated with impaired neuromuscular transmission in a group of patients with limb-girdle myasthenia Aim: To identify structural and functional abnormalities of the NMJ and whether the defects are pre- or post-synaptic. To try to identify a genetic dysfunction in components of the NMJ. Methods: Electromyography was used to investigate NMJ transmission in vivo. Muscle biopsies were taken from patients for intracellular recordings to be obtained in vitro. Light and electron microscopy were used to identify morphological changes in NMJs of LGS patients. Immunocytochemistry was used for protein localisation studies. Genomic DNA was obtained from patients for DNA analysis. Results and Conclusions: Though quantal content and AChR number was 50% lower than controls, the contact area of the NMJ and the amount of postsynaptic folding was also 50% lower, so the quantal content per unit area of synaptic content and the AChR density was similar to controls. Immunocytochemistry showed that distributions of the proteins investigated were as normal. DNA analysis did not find any mutations in the NMJ proteins investigated. It seems that impaired NMJ transmission is due to structural abnormalities rather than the neurochemical process of signalling. This highlights the importance of understanding the mechanisms of development of the NMJ, and how the proteins involved in this may be impaired. Whilst the patients seemed to share similar syndromes, there does not as yet seem to be a clear identified defining feature that is unique to LMS.

4 Ac#ve Zones and the Readily Releasable Pool of Synap#c Vesicles at the Neuromuscular Junc#on of the Mouse Background Informa#on NeurotransmiCer release during synap#c transmission is highly regulated. The synchronous release of synap#c vesicles takes place at specialised areas of the presynap#c membrane called ACTIVE ZONES (AZs).

5 Background Informa#on Previous research has described the gross structure of AZs, demonstra#ng that they vary from species to species and even at different synapses within the same species. The number of AZs at each synapse also varies considerably throughout the nervous system. Background Informa#on We do not yet understand en#rely the complex processes involved in quantal release and vesicle replenishment at AZs. We know that in the mouse there are two docked vesicles per AZ with a number of nearby secondary docked vesicles and others that are held nearby by tethering to filaments from the AZ.

6 Aims and Hypothesis of the Paper This paper aims to characterise the dynamics of the RRP of neurotransmicer vesicles at AZs in mouse motor terminals. Aims and Hypothesis of the Paper The authors intend to es#mate the size of the RRP by extrapola#ng from morphological data and then to confirm their es#mate using electrophysiological data. They then intend to explore the effects of various physical factors on the dynamics of vesicle exocytosis and replenishment.

7 Methods The levator auris longus muscles were dissected from mice and prepared in a physiological solu#on at room temperature. The nerves were s#mulated using a suc#on electrode while mepps and EPPs were recorded. μ conotoxin was used in order to block skeletal muscle voltage gated sodium channels. Methods Quantum contents during at train of impulses were recorded and ploced against #me. The authors hypothesised a mathema#cal model to describe the pacern they saw.

8 Methods Immunohistochemistry was used to image AZs with an#bodies against Bassoon and Piccolo which are scaffold proteins enriched at AZs. Morphological Results Figure A shows the method for coun#ng AZ spots. Increasing segmenta#on. They counted the number of spots and calculated average width of them and their results correlated well with those of previous freeze fracture results (Fukunaga et al )

9 Morphological Results The authors found that the spots were sta#s#cally evenly separated as the appear by eye. They formulated random arrays of spots in order to show that this finding wasn t by chance and their results confirmed that the even spacing was significant. Morphological Results By marking the centre of the Bassoon spots and comparing their loca#on to AChRs marked by BTX Rho on the postsynap#c membrane, it was shown that the AZs appeared to lie directly over corresponding postsynap#c folds.

10 Morphological Results High level s#mula#on did not disrupt the pacern of spot staining, demonstra#ng that AZs are very stable. Treatment with proteoly#c enzyme disrupted the pacern. These results are taken in conjunc#on to prove that Bassoon staining does indeed highlight AZs. Electrophysiological Results Having demonstrated that there are ~850 AZs per terminal and assuming that there are two docked vesicles per AZ the RRP of docked and primed vesicles was calculated to be 1700.

11 Electrophysiological Results These graphs show: A Peak EPP amplitudes during 1000 ms of 100 Hz s#mula#on. B Quantum Content decrease and plateau over #me. C Ini#al method of es#ma#ng the RRP rejected. D and E Further methods for es#ma#ng the RRP. E takes into account the kine#c proper#es of the recruitment process as well as the synchronicity of release and replenishment. Electrophysiological Results The average size of the RRP calculated by the third method was 1730 ± 55 quanta. According to this model, during the first ~200ms of s#mula#on roughly 90% of quanta come from the RRP and aier s#mula#ons at 100Hz almost all the quanta from the ini#al RRP have been released. The pool decreased for ~50ms and then rose briefly before declining to a plateau level. This biphasic ac#on has been described before in skeletal muscle whereby there is some#mes a slight mismatch between vesicle exocytosis and endocytosis rates (Elmqvist and Quastel, 1965).

12 Electrophysiological Results Next an es#mate of the rate of RRP refilling was calculated by giving pairs of s#mula#ons separated by varying #me intervals and using the previous method for calcula#ng RRP size. Electrophysiological Results E shows that the apparent size of the RRP was not constant with frequency of s#mula#on, but more than doubled as the frequency increased from 1 to 100 Hz. One interpreta#on of this result is that not all AZs are func#onal at low s#mulus frequencies, but all are recruited during high frequency s#mula#on, presumably because of the rise in global calcium, the rate of which is rapid.

13 Electrophysiological Results Conclusions The paper greatly improves the previously rather basic understanding of the kine#cs of the RRP at AZs: we learn an approximate size of the RRP, and it s kine#cs of exocytosis and refilling in response to varying frequencies of s#mula#on, temperatures and chemicals. There is a good use of quan#ta#ve data to back up conclusions. There are quite a lot of graphs, but they are well explained and clear and greatly aid the reader in understanding the discussion in the text.

14 Conclusions The significance of phorbol ester and EGTA is not explained. The authors say that they inves#gated a number of different models to extrapolate RRP size from s#mula#on data, but don t make clear what these other models were. They only present one model that happens to agree with their predic#on based on morphological data. They do not concede that the kine#cs seen may vary between different synapses in the same animal and between animal species. Natalia Petrova

15 Hypothesis: Postsynaptic folds and the VGSCs within them contribute to the safety factor for neuromuscular transmission Aim: Investigation of the effect of this contribution and estimation of the ratio of the normal quantal content to the number of quanta required to reach threshold (safety factor) in fast and slow-twitch muscles Muscles: Extensor Digitorum Longus (EDL) muscle, fast-twitch Soleus muscle, short-twitch

16 Methods and Results Normal quantal content was measured in both muscles by blocking APs using μ-conotoxin. EPPs, MEEP, EPCs and MEPCs were recorded. The number of quanta required to reach threshold was determined in muscles incubated with d-tubocurarine (dtc). APs were generated in both junctional (J) and extra-junctional (XJ) regions.

17 Quantal Content Measurement Higher MEPP frequency for EDL Mean amplitude of nerve-evoked EPCs in EDL is 60% larger Measurement of the quantal content is 30-40% higher in EDL Threshold Measurement EDL shows a higher amplitude of threshold EPCs No significant different between the two muscles in the amount of charge required to reach threshold Both muscles require approximately 13 quanta to reach threshold

18 The depolarisation required to reach threshold was lower at the J than at the XJ of regions in the soleus muscle. The amount of charge required to reach threshold in J is approximately 20% lower than XJ in both muscles

19 Safety factor calculation in J, XJ and nerve evoked stimulation, in response to current and voltage Nerve-evoked excitation reveals a safety factor of 3.5 in the soleus muscle and 5 in the EDL Safety factor for nerve evoked excitation is about twice the value of that in XJ region Amount of membrane per fold is the same in both muscles Synaptic area of EDL is 64% of that in soleus EDL releases more than twice as much transmitter per unit area of synaptic contact

20 Discussion The safety factor of nerve-evoked stimulation is 3.5 and 5 in the soleus and EDL muscle fibres respectively Ratio N/XJ is about 2 in both Hypothesis confirmed Experimental conditions vs. In vivo Limitations Future study: Comparison between different muscle fibre types Future Reading Myasthenia gravis: past, present, and future Bianca M. Conti-Fine, Monica Milani and Henry J. Kaminski Molecular Architecture of the Neuromuscular Junction Benjamin W. Hughes, PhD, Linda L. Kusner, PhD, and Henry J. Kaminski, MD

21 Severely impaired neuromuscular synap#c transmission causes muscle weakness in the CACNA1A mutant mouse rolling Nagoya Simon kaja, Rob. C.G. Van de Ven, J. Gert van Dijk, Jan J. G. M. Verschuuren, Kiichi Arahata, Rune R. Framts, Michel D. Ferrari, Arn M. J. M. Van den Maagdenberg and Jaap J. Plomp European Journal of neuroscience, Vol. 25, pp , 2007 Aim to inves#gate the NMJ of RN mice with in vivo and ex vivo electrophysiology methods. Mutant mouse rolling Nagoya a neurological mutant which exhibits sever ataxia. Found due to a recessive point muta#on in the Cacna1a gene Causes an amino acid change in the voltage sensing S4 segment of the pore forming subunit of the Cav2.1 voltage gated Ca2+ channels.

22 Methods in vivo Grip strength assessment 5 trials 30 seconds apart, peak force recorded. Inverted grid hanging test 3 trials 30 seconds apart, average hanging #mes calculated. Repe##ve nerve s#mula#on electromyography (RNS EMG) of the compound muscle ac#on poten#al from foot muscles. Trains of 10 s#muli at increasing frequencies, 2 minutes between trains. CMAP amplitude and area of ini#al nega#ve peak were measured for all the CMAPs in a train Methods ex vivo Mice killed by carbon dioxide inhala#on Muscle contrac#on experiments Phrenic nerve hemidiaphragm Tetanic contrac#on force recorded Increasing concentra#ons of d tubocurarine NMJ electrophysiology Phrenic nerve hemidiaphragms, soleus and flexor digitorum brevis muscles Recordings of MEPPS and EPPS were made at the NMJ Quantal content was calculated at each NMJ Also measured in presence of ω agatoxin, a specific Cav2.1 channel blocker

23 Results muscle weakness and fa#gue A mean pulling force was reduced to 38% of wildtype in RN mutant mice. B 4 of 5 normal mice managed to reach up to 5minutes hanging #me whereas the RN mice scored #mes from 7 16seconds C the first CMAP amplitude was also averagely 50% smaller in RN mice than Wildtype CMAP amplitudes decreased much faster in RN mice(74%) than wildtype (7.1%). Reduced safety factor of transmission at the RN NMJ A shows how the tetanic contrac#on force is effected more greatly within RN mice than wildtype when in the presence of dtubocurarine RN muscles are more sensi#ve to dtubocurarine

24 Reduced evoked transmicer release B shows the amplitude of the EPP s#mulated, can see they were 40% smaller in RN mutants. C shows the quantal content released during an EPP, it is again greatly reduced in the RN mutant. The ω agatoxin is included to show the results are due to the Cav2.1 channel and thus RN muta#on. Increased spontaneous ACh release A increased spontaneous ACh releas ω anatoxin reduces the frequency of MEPPs in all genotypes shoing that the MEPPS are due to the Cav2.1 channels and an effect of the RN muta#on.

25 Increased spontaneous ACh release The MEPP was also recorded in the flexor digitorum brevis and soleus muscle where again the frequency was seen to be greatly increased. Conclusions RN mutants suffer from muscle weakness and fa#gability Reduced safety factor of neuromuscular transmission within RN mutants Increased spontaneous ACh release and decreased evoked transmicer release both which are due to the mutated Cav2.1 calcium channel

26 Big burning ques#ons How similar is this RN mouse model to LEMS? Are the clinical effects(eg ataxia) a detractor? How are MEPPS/EPPs co regulated in this model? PRE AND POST SYNAPTIC ABNORMALITIES ASSOCIATED WITH IMPAIRED NEUROMUSCULAR TRANSMISSION IN A GROUP OF PATIENTS WITH LIMB GIRDLE MYASTHENIA C. R. Slater, P. R. W. FawceC, T. J. Walls, P. R. Lyons, S. J. Bailey, D. Beeson, C. Young and D. Gardner Medwin

27 Ini#al Aim to determine the underlying defects in neuromuscular transmission and whether they are due to structural or func#onal abnormali#es Determine whether the defects that account for impaired neuromuscular transmission in LGM pa#ents are pre or post synap#c Major Findings Main conclusion: impaired NMJ transmission in these LGM pa#ents results from structural abnormali#es including reduced size and postsynap#c folding, rather than any abnormality in the immediate events of transmission Both sides of the synapse are involved.

28 Clinical Manifesta#ons Onset in infancy or childhood Weakness primarily of the proximal muscles Fluctua#ons of the severity on a #mescale of weeks. Progressed from moderate to severe over several years. Characteris#c sinuous or waddling gait Methods 8 pa#ents with common symptoms that varied from the diagnos#c features of other known congenital myasthe#c syndromes, MG and LEMS Cellular, subcellular and molecular detailed studies of each pa#ent including a motor point muscle biopsy for diagnosis of detailed mechanisms for LGM group and control group Control group pa#ents with idiopathic muscle pain or adult onset muscular dystrophy and no other obvious neurological involvement

29 Electromyographic Studies CMAP compound muscle ac2on poten2al Trains of 8 10 s#muli at 3Hz delivered at rest, at intervals and aier 20s maximal voluntary contrac#on A shows decrement in CMAP at rest is abolished 10s aier 20s of ac#vity but returns 2 mins later. B administra#on of edrophonium increases CMAP amplitude at 30s and reduces decrement 1 min later. Single fibre EMG C JiCer (the trial to trail varia#on in the latency from s#mulus to response) is increased and intermicent failure of excita#on of muscle fibres block. mepps, EPPs and mepcs and EPCs were measured. Measured amplitude and exponen#al decay #me constant for both. Evoked (EPP, EPC) but not spontaneous (mepp, mepc) synap#c responses are reduced in LGM pa#ents

30 Light Microscopy, appearance of NMJs in LGM and Control. A, B motor axon terminals. C, D AChs E,F AChE ac#vity Electron Microscopy A, B Control, abundant postsynaptic folds C, D LGM, greatly reduced folding

31 Overview of Findings CMAP compound muscle ac#on poten#al decrement on repeta#ve s#mula#on at 3Hz SFEMG single fibre EMG increased jicer and blocking confirming impaired transmission No serum an#bodies to AchRs Intracellular recordings quantal content, area of synap#c contact and postsynap#c folding and no. of AChRs per NMJ all 50% reduced. Immunolabelling studies normal distribu#on and abundance of NMJ proteins. DNA analysis no muta#ons were found in in the genes encoding AChR subunits, rapsyn, ColQ, ChAT or Muscle Specific Kinase. Treatment Treatment is symptoma#c Pyridos#gmine Some showed long term improvement Some deteriorated with treatment Some had intolerable side effects 3,4 diaminopyridine Some showed transient improvement Ephedrine Helpful in half of pa#ents

32 Reduced size of NMJs Main Findings Reduced post synap#c folding Area of synap#c contact and quantal content reduced by 50% Quantal release per unit area, size and kine#cs of mepcs and protein distribu#on normal Conclusion: abnormality in the mechanisms that determine NMJ size and confirma#on What Happened Next Further conclusion: regulatory events influencing the structure and maintenance of the NMJ account for impaired neuromuscular transmission in LGM pa#ents This study may not have found the cause to LMG but the findings stated here allowed the elimina#on of several possible candidates. DOK7 muta#ons! Dok 7 (gene DOK7) binds to MuSK and plays a role in amplifying signalling to downstream pathways that control clustering of AChRs on the postsynap#c membrane.