Dynamic remodelling of synapses can occur in the absence of the parent cell body
© Bettini et al; licensee BioMed Central Ltd. 2007
Received: 08 February 2007
Accepted: 26 September 2007
Published: 26 September 2007
Retraction of nerve terminals is a characteristic feature of development, injury and insult and may herald many neurodegenerative diseases. Although morphological events have been well characterized, we know relatively little about the nature of the underlying cellular machinery. Evidence suggests a strong local component in determining which neuronal branches and synapses are lost, but a greater understanding of this basic neurological process is required. Here we test the hypothesis that nerve terminals are semi-autonomous and able to rapidly respond to local stimuli in the absence of communication with their parent cell body.
We used an isolated preparation consisting of distal peripheral nerve stumps, associated nerve terminals and post-synaptic muscle fibres, maintained in-vitro for up to 3 hrs. In this system synapses are intact but the presynaptic nerve terminal is disconnected from its cell soma. In control preparations synapses were stable for extended periods and did not undergo Wallerian degneration. In contrast, addition of purines triggers rapid changes at synapses. Using fluorescence and electron microscopy we observe ultrastructural and gross morphological events consistent with nerve terminal retraction. We find no evidence of Wallerian or Wallerian-like degeneration in these preparations. Pharmacological experiments implicate pre-synaptic P2X7 receptor subunits as key mediators of these events.
The data presented suggest; first that isolated nerve terminals are able to regulate connectivity independent of signals from the cell body, second that synapses exist in a dynamic state, poised to shift from stability to loss by activating intrinsic mechanisms and molecules, and third that local purines acting at purinergic receptors can trigger these events. A role for ATP receptors in this is not surprising since they are frequently activated during cellular injury, when adenosine tri-phosphate is released from damaged cells. Local control demands that the elements necessary to drive retraction are constitutively present. We hypothesize that pre-existing scaffolds of molecular motors and cytoskeletal proteins could provide the dynamism required to drive such structural changes in nerve terminals in the absence of the cell body.
Retraction of nerve terminals is a fundamental event in the development and maintenance of the nervous system [1, 2], occurring when exuberant neuronal projections are pared back by activity-dependent processes. These events have been extensively studied at the neuromuscular junction (NMJ) during development [3, 4], re-innervation  and following axotomy . Here, competing nerve terminals at muscle motor endplates are withdrawn bouton-by-bouton in an apparently controlled, piecemeal fashion until the last bouton withers and is re-absorbed into the parent axon [4, 7, 8]. However, relatively little is known about the cellular mechanisms that govern progressive nerve terminal retraction. This information is particularly pertinent since nerve terminal loss is emerging as a key early event in many neurodegenerative diseases traditionally thought of as disorders of cell bodies [9–12]. In fact nerve terminal loss has now been observed as the earliest event in spinal muscular atrophy [13, 14], progressive motor neuropathy [15, 16], amyotrophic lateral sclerosis , motor neurone degeneration and SOD1 over expression . Additionally, in the CNS Huntington's  and Alzheimer's  diseases both show early nerve terminal loss.
Available data points to a degree of synaptic independence in the retraction of individual synaptic connections. Observations of single motor units during developmental synapse elimination reveal three quite separate populations of nerve terminals: stable, actively withdrawing and actively enlarging [3, 12] and it is difficult to envisage how a single cell soma could directly co-ordinate this range of actions in different parts of its terminal arbour. Adult axons and terminals appear to contain little if any machinery for protein synthesis, but the first signs of regenerative sprouting occurs within a day of axotomy [20, 21], which appears too rapid for communication from the site of injury to the cell body and back, even by fast axonal transport. Neonatal synapse elimination can proceed subsequent to axotomy  and co-ordinated structural changes at presynaptic nerve terminals (retraction) can occur in the absence of parent cell somas. Axons disconnected from their cell bodies in vitro can assemble new growth cones at lesion sites [22–24] and transected axons are able to mount a regenerative response in the absence of cell somas . Taken together these data suggest that the machinery necessary to drive synapse loss (and re-growth) may be constitutively present in the nerve terminal and axon and that communication with the cell body is unnecessary. Although other evidence, primarily from the hippocampus, demonstrates that long-term alterations in synaptic strength at subsets of neurones in a synaptic field may be driven by pre-existing or newly synthesised plasticity related proteins which differentially bind to synapses dependent upon their level of activity. This is thought to be regulated by synaptic tagging , where single synapses can show independent behaviour.
In this study we show that nerve terminal retraction can be triggered in the absence of neuronal cell bodies by the extracellular application of an ATP analogue. The nature of the nerve terminal retraction does not resemble Wallerian or Wallerian-like degeneration; rather it resembles 'dying back' neuropathies and synapse elimination.
BzATP triggers the rapid loss of nerve terminals in the absence of cell bodies
BzATP acts at presynaptic purinoceptors to trigger nerve terminal loss
ATP acts at a range of purinoceptors, though our previous studies have only been able to demonstrate P2X7 receptor subunits (P2X7RS) on motor nerve terminals . However, there is evidence for the presence of P2Y receptor subunits on Schwann cells  and muscle fibres . We therefore further investigated the possible involvement of purinoceptors in the BzATP-induced response with selective pharmacological agents. Pre-incubation with a selective antagonist for P2X7RS, Brilliant blue G (BBG: 1 μM) either partially blocked or slowed BzATP-induced nerve terminal loss (occupied, 74 ± 4%; intermediate, 25 ± 5%; unoccupied, 0%, N = 3, n = 300). Reactive Blue 2 (100 μM), which is reported to block P2Y and P2X7 receptor subunits, completely blocked BzATP-induced nerve terminal retraction (occupied, 100%, N = 3, n = 300). Complete block of BzATP-induced retraction was also obtained with the broad-spectrum P2 antagonist Suramin (100 μM: occupied, 100%, N = 2, n = 200). Quantitative data from these experiments are summarized in Figure 2.
Remaining axons and nerve terminals transmit action potentials and recycle neurotransmitter
Early ultrastructural events at neuromuscular junctions are consistent with nerve terminal retraction
In this study we demonstrate that in preparations where distal nerve axons, terminals and associated skeletal muscle fibres, BzATP can trigger the retraction of nerve terminals from post-synaptic muscle fibre endplates even though they are no longer connected to their parent cell bodies. The events triggered most closely resemble retraction events during neuromuscular synapse elimination. This action of BzATP appears to be mediated by presynaptic P2X7 receptors.
Isolated axons and terminals can undergo dynamic cellular re-arrangements
In these experiments we show that a brief pharmacological signal can trigger rapid and significant morphological events in cell fragments consisting of isolated nerve terminals and attached distal axon stumps. We suggest that the morphology of these events most closely resembles nerve terminal retraction during developmental synapse elimination [4, 6, 33, 36], following nerve section in the slow Wallerian degeneration mouse  and in motor neurone disease . We also observed fragmentation of pre and/or postsynaptic sites at a small number of neuromuscular junctions and interestingly similar synaptic responses have also been observed during developmental synapse elimination  and in mouse models of spinal muscular atrophy . Therefore we suggest that the events seen here are part of the normal physiological/pathophysiological repertoire of neurones, which have been triggered by activation of pre-synaptic purinergic receptors. It will be interesting to determine the exact rate and timecourse of the events described here by using confocal and timelapse imaging. However, this may not in fact further illuminate the underlying molecules and mechanisms at play. Significantly, during the course of these studies we found no evidence of gross, injury-induced degenerative events up to and including 5 hr after axons were cut during dissection. This agrees with our previous findings which indicated that Wallerian degeneration does not begin until at least 12 hours after dissection . Specifically, we saw no evidence of terminal Schwann cells engulfing degenerating nerve terminal boutons in-situ, mitochondrial degradation or cytoplasmic vacuolation [32, 38], which would indicate degenerative events. We did however, see partially occupied 'intermediate' junctions (Fig 1c, d) and intact nerve terminal boutons which had apparently withdrawn from post-synaptic sites (Fig 5c), both of which are atypical of degenerative changes, and characteristic of retraction events. Occasionally we found evidence of fragmented or absent muscle endplates, associated with intact or fragmented nerve terminals. As P2X2 receptor subunits have recently been shown to have a role in the formation of neuromuscular junctions , it is possible that this indicates a direct effect on post-synaptic structures or nerve to muscle communication pathways.
Several other groups have described dramatic morphological events at the neuromuscular junction subsequent to insult. Is it likely then that the events described here are simply a response to toxic insult? Addition of a component of Black Widow Spider venom, α-latrotoxin , triggers massive exocytosis of vesicles which produce presynaptic 'arches', similar in appearance to the mega-omega profiles described here. However, these junctions recover and do not progress to disconnection of nerve terminals from muscle fibre membranes or insertion of Schwann cell membranes into synaptic clefts. Blocking of axoplasmic flow by the addition of colchicine  after a period of days leads to an increased density of synaptic vesicles, mitochondrial disruption, retraction and Schwann cell wrapping of nerve terminal boutons. However, we did not see mitochondrial damage or obvious signs of synaptic vesicle accumulation, if anything vesicles were slightly depleted compared to control preparations. In addition, colchicine produces gross changes in the post-synaptic muscle fibre membrane associated with the loss of junctional folds as the nerve terminal withdraws. Complement-mediated anti-ganglioside attack  produces electron lucent nerve terminals with damaged mitochondria, invasion of Schwann cells into the synaptic cleft and wrapping of nerve terminal boutons. We did not see evidence of such degenerative changes, though we did observe similar insertion and wrapping of nerve terminal boutons by Schwann cell processes, but this is also a common feature of synapse elimination [33, 36, 37]. All of these situations have disruption of mitochondria as a significant, and often early event, which we did not observe following treatment with BzATP. Further, nerve terminals exhibited selective vulnerability to the BzATP pulse, which is not the case in toxic injury. We would predict that similar events could be demonstrated in vivo and on non-axotomised nerve terminals , and this is an avenue which will be pursued in future studies.
Activation of P2 receptors trigger nerve terminal loss
We show that a stable analogue of ATP triggers structural changes in nerve terminals. BzATP is relatively selective for P2X7RS, but more importantly this is so far the only member of the ionotropic family of P2X receptors reported on motor nerve terminals [28, 29]. P2X receptors have however, been described on muscle fibres  and Schwann cells . The validity of tools used to demonstrate P2X7RS on neurones have been questioned, but the current consensus is that the original reports  are accurate . We suggest that BzATP acts, at least in part, on P2X7RS. BzATP-induced nerve terminal retraction was incompletely prevented by the P2X7RS selective blocker BBG, but completely prevented by RB2 and Suramin, which block both P2X and P2Y receptor, subtypes. Therefore it is likely that BzATP also targets P2Y receptors in these experiments. We do not know to what extent P2X and P2Y receptors work via common or separate signalling pathways and currently available drugs are not sufficiently selective to allow us to unravel the relative involvement of these sub-families at the present time. Interestingly, P2Y receptors have been reported on both Schwann cells  and muscle fibres , which could provide potential pathways for the observations of Schwann cell invasion and endplate dismantling. Activation of P2X7RS can open a cation-permeable ionotropic channel or a large (membrane permeating) pore . We have previously demonstrated that BzATP does not open a large pore at motor nerve terminals by simultaneous incubation in a solution of the membrane-impermeant fluorochrome, 6-carboxyfluoroscein (see figure 5 in ). This suggests that if activation of P2X7RS is a key event in this response, it is the ionotropic channel and not the membrane permeabilising large pore that is responsible.
When might ATP rise to sufficient levels to trigger these responses in vivo? First, following direct muscle trauma when loss of membrane integrity leads to the release of muscle sarcoplasm containing approximately 8 mM ATP  into the extracellular space. Second, during tissue ischaemia when both neurones and muscle fibres release ATP [49, 50] and ionic gradients across cellular membranes collapse resulting in a fall in the concentrations of extracellular divalent cations . This combination of factors facilitates P2X7RS activation by removal of a resting cation-block [43, 52]. One other group have described what appear to be similar retraction events following ischaemia in a preliminary report , and another have implicated P2X7RS in hypoxic damage at synapses in the CNS , hinting that ATP could be a key factor in hypoxic damage. It is interesting to note that P2 receptor antagonists improve recovery after spinal cord injury , pointing to a possible general mechanism for dynamic rearrangement of synapses through nerve terminal loss following chronic or acute injury.
Autonomous events in nerve terminals
We have demonstrated that BzATP triggers a controlled retraction of nerve terminals from post-synaptic sites. Importantly, this occurs in isolated nerve-muscle preparations where all connectivity with the parent motor neurone cell bodies has been abolished. The heterogeneity of synaptic responses recorded is similar to events described in single motor units during developmental synapse elimination [4, 35] and following axotomy in mutant slow Wallerian degenerating (Wlds) mice . These observations, taken together with other data [4, 6], support the notion that nerve terminals are autonomous or 'compartmentalised' , each compartment containing specific elements/processes enabling them to respond in different ways to environmental signals. If local, compartmentalised processes are important in retraction events, how are they controlled? Changes in cell shape require alterations to the cytoskeleton and it seems unlikely that new cytoskeletal elements could be generated and/or assembled within the time frame of these experiments, especially in the absence of neuronal cell bodies. An attractive hypothesis therefore is that sufficient cytoskeletal elements to drive nerve terminal retraction are constitutively present in distal axons and terminals and that re-arrangement of these drive retraction events. Non-muscle myosins IIA and IIB drive neurite elongation and retraction in culture [57, 58] and we have previously shown that these are present in motor nerve terminals . Here a pre-assembled 'molecular clutch'  regulated by the differential activation of opposing myosin motors by kinases could drive cytoskeletal rearrangement, and therefore morphological change, without the requirement for generation of new cytoskeletal elements. It remains unclear how purinergic receptors might be linked to the cytoskeleton, but evidence exists to link similar pathways in other systems. Extracellular purines acting on membrane-bound receptors trigger dissagregation of the actin cytoskeleton in the WRK-1 (mouse mammary tumour) cell-line. This may be related to differential activation of molecular motors . Proteomic analysis of the P2X7R indicates several cytoskeletal elements present within the receptor complex  and one of these, supervillin, binds to actin and non-muscle myosin II. Supervillin is an important adapter protein in the organisation of attachments of the cytoskeleton to dynamic regions of membranes  which could drive shape changes at nerve terminals.
In summary, we provide evidence that following disconnection from their parent cell bodies, activation of P2X7 receptors on nerve terminals by BzATP triggers synaptic retraction. This retraction closely resembles similar events occurring during synapse elimination and motor neurone disease. We suggest that molecules and mechanisms constitutively present in the distal axon and terminal are sufficient to drive this retraction response. Further, we speculate that signals from the parent cell soma may not be necessary to control retraction during synaptic plasticity.
Adult C57BL/6 mice were killed by CO2 intoxication, and intact preparations of lumbrical muscles, plantaris tendon and sciatic/tibial nerves were dissected and pinned out at resting length in Sylgard-lined Petri dishes containing oxygenated (95%O2/5%CO2), calcium and magnesium free physiological saline (122.4 mM NaCl, 5 mM KCl, 0.4 mM NaH2PO4, 23.8 mM NaHCO3, 5.6 mM Glucose, 5.5 mM Hepes, pH 7.2–7.4) at room temperature. These were transferred to oxygenated physiological saline as above, and any muscles which failed to twitch in response to nerve stimulation were excluded from further assay. Preparations were either maintained throughout in oxygenated physiological saline or alternatively in the same saline supplemented with several different purinergic agonist and antagonists. Purine receptor agonists and antagonists used were as follows: 2'-3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP: 100 μM), Brilliant blue G (BBG: 1 μM), Reactive Blue 2 (RB2: 100 μM), Suramin (100 μM). All experiments began within 60 min of animal sacrifice.
After the experimental period (3 hrs), preparations were fixed in 4% paraformaldehyde for 30 mins and stained for immunofluorescence microscopy. Synaptic vesicles and neurofilaments were labelled with a combination of SV2 (1:500) and NF165 (1:250, developed by T.M. Jessel and J. Dodd, and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, USA), and visualised with Cy2- (1:200, donkey anti mouse: Jackson laboratories, USA) secondary antibody. Post-synaptic acetylcholine receptors were labelled with tetramethyl rhodamine isothiocyanate conjugated ãbungarotoxin (5 μg/ml, TRITC-a-BTX: Molecular Probes, Leiden). Slides were coverslipped in 2.5% n-propyl gallate in glycerol and viewed with epifluorescence illumination. In preliminary experiments we determined that a 15 min pulse of BzATP was the optimum trigger for nerve terminal retraction.
Mice were killed and handled as above, but in this case intact preparations of flexor digitorum brevis muscles and associated sciatic/tibial nerve were dissected and placed Immediately after dissection into freshly prepared and oxygenated physiological saline. Addition of chemicals began within 60 minutes of animal sacrifice. BzATP (100 μM) was added for 30 min and preparations were fixed in buffered 4% paraformaldehyde/0.2% glutaraldehyde, pH 7.2 either immediately or subsequent to a 60 min washout period. Muscles were fixed and labelled for electron microscopic localisation of P2X7 receptor subunits (P2X7RS) as previously described .
Preparations were treated identically to those above, except at the end of the 3 hr period, they were vitally labeled and imaged. Preparations were loaded with the vital styryl dye RH414 (10 μg/ml; Molecular Probes) in physiological saline solution by nerve stimulation (suprathreshold 0.1 msec pulse trains delivered at 10 Hz for 10 min to the intercostal nerve via asuction electrode), followed by at least 1 hr washing in oxygenated physiological saline. Preparations were transferred to the microscope stage and terminals were imaged.
25 sequentially identified endplates from randomly oriented immunostained lumbrical muscles were identified and quantified for the degree of congruence between overlying nerve terminal and muscle endplate from four muscles per hindfoot. Three categories were used; 'occupied', denoting complete occupancy; 'intermediate', where either endplate was partially occupied by nerve terminal or was fragmented; 'unoccupied', where no nerve terminal was present at the endplate. Occasionally, damaged muscle fibres or unusually deep endplates were difficult to accurately assess, and were discarded from the data set. Data are presented as mean ± SEM, N = number of mice, n = number of endplates. GraphPad Prism (Graphpad software) was used to carry out statistical tests.
NLB was supported by a Wellcome Trust Value in People Award, TSM was a Physiological Society Vacation Scholar, SHP and BB are supported by the Anatomical Society of Great Britain and Ireland. JD is supported by The British Heart Foundation and The Wellcome Trust. Thanks to Professor Richard Ribchester and Dr Tom Gillingwater for helpful comments on the manuscript.
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